Difference between revisions of "Team:Stanford-Brown/SB16 Protocols"

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<title>Amide Synthesis with trimethyl borate · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv780-title" data-index="0">Amide Synthesis with trimethyl borate</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv780-intro" data-index="0">Synthesis of amide bonds with trimethyl borate as the coupling reagent. Used to produce polymers from p-aminobenzoic acid monomers.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m781" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c782" data-index="0,0">2 equiv. P-aminobenzoic acid (pABA)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c783" data-index="0,1"><span style="color:#344043"><span style="background-color:#FFFFFF">2 equiv. trimethyl borate</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c784" data-index="0,2">Nonpolar/aprotic solvent (MeCN)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c785" data-index="0,3"></div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s786" data-index="0">Amide synthesis</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s786" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s787" data-index="1">Add pABA (1 equiv) to MeCN (2 mL, 0.5 M)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s787" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s788" data-index="2">Add 1 equiv trimethyl borate to mixture. Stir at 80C (100C?) for 5-24 hours.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s788" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s789" data-index="3">Solid Extraction</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s789" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s790" data-index="4">Dilute reaction mixture with CH2Cl2 or EtOAc (organic solvent) and H2O (polar solvent).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s790" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s791" data-index="5">Add MgSO4 (anhydrous) to remove H2O.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s791" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s792" data-index="6"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s792" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
  
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<meta charset="utf-8"/>
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<title>Annealing of Template DNA · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv696-title" data-index="0">Annealing of Template DNA</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv696-intro" data-index="0">Use this protocol to anneal complimentary single stranded oligonucleotides.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m697" data-index="0">Measurements per Tube:</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c698" data-index="0,0">Forward Seq (200 µM)              10 µl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c699" data-index="0,1">Reverse Seq (200 µM)              10 µl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c700" data-index="0,2">10x H Buffer                                2 µl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c701" data-index="0,3">D.I. Water                                    28 µl</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m702" data-index="1">Total Volume per Tube:      50 µl</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m703" data-index="2">Final Concentration:          80 mM</div><ul class="components"></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s704" data-index="0">Anneal Strands</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s704" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s705" data-index="1">Heat mixed tubes at 93°C for 1 min in thermal cycler</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s705" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s706" data-index="2">Decrease heat of thermal cycler by 5°C every 2 min with small ∆T/sec in between steps</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s706" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s707" data-index="3">When the temp falls more than 20°C below the Tm of the strands to anneal, change steps to decreasing 10°C every min</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s708" data-index="4">Once at room temperature, hold samples at 4°C for 15 min, then store samples at -30°C</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s708" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
  
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<meta charset="utf-8"/>
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<title>BCA Protein Assay · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv605-title" data-index="0">BCA Protein Assay</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv605-intro" data-index="0">For determining concentration of protein using Nanodrop. summarized from http://www.nanodrop.com/Library/T085-NanoDrop-2000-2000c-BCA%20Protocol.pdf
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m606" data-index="0">Working reagent (<span style="color:#344043"><span style="background-color:#FFFFFF">50A:1B)</span></span></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c607" data-index="0,0">Pierce BCA Protein Assay Reagent A</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c608" data-index="0,1">Pierce BCA Protein Assay Reagent B</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m609" data-index="1">BSA/Albumin Standard (2mg/mL)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m610" data-index="2">PCR tubes/PCR plate</div><ul class="components"></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s611" data-index="0">Sample Prep</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s611" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s612" data-index="1">Let all reagents/samples/standards equilibrate to RT.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s612" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s613" data-index="2">Prepare enough fresh working reagent (WR) for all standards and samples to be measured using a <b>50:1 ratio</b> of the kit reagents A:B. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s613" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s614" data-index="3">If using high-range assay for 5 samples, you'll need approximately 1000 uL of WR total ==&gt; So, you'll need about 1000 uL of A and 20 uL of B (ratio would be 1000:20).</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s615" data-index="4">Add reagent volume to each PCR tube.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s615" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s616" data-index="5">Microassay (1:1 sample to WR) Add 10 uL of WR to each standards and sample tube/well.</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s617" data-index="6">High range assay (1:20 sample to WR) Add 200 uL of WR to each standards and sample tube/well.</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s618" data-index="7">Add 10 uL of standards or samples to the appropriate tube. Mix well by gentle vortexing.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s618" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s619" data-index="8">Incubate standard and sample tubes at:</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s619" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s620" data-index="9">37 C for 30 min OR 60 C for ~5 min. Cool to room temp after.</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s621" data-index="10"></div></div></li></ul></div></div></div></div></div></div></div></div></div></div></div>
  
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<title>Cleaning Electrophoresis Gel · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv874-title" data-index="0">Cleaning Electrophoresis Gel</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv874-intro" data-index="0">Cleaning electrophoresis gel with gel extraction kit (purple).
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m875" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c876" data-index="0,0">100% ethanol</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c877" data-index="0,1">WN + WS buffers</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c878" data-index="0,2"></div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s879" data-index="0">Gel Cleaning</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s879" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s880" data-index="1">Prepare WN + WS buffers by adding appropriate amount of 100% ethanol as shown on bottle.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s880" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s881" data-index="2">Carefully cut out piece of gel containing fragment of interest under longwave UV.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s881" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s882" data-index="3">Add 300ul GEX for every 100mg gel collected to centrifuge tube and gel piece.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s882" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s883" data-index="4">Incubate at 55˚C for 5-10min until gel has dissolved. Can smash with a pipette tip.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s883" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s884" data-index="5">Place extraction column into collection tube and load 0.7ul mixture into column. Centrifuge at 5000rpm for 30s. Discard flow-through. Repeat until all mixture is used.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s884" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s885" data-index="6">Wash column with 0.5ml (500ul) of WN buffer by centrifuging at 5000rpm for 30s. Discard flow-through.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s885" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s886" data-index="7">Wash column with 0.5ml WS buffer by centrifuging for 60s at 5000rpm. Discard flow-through.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s886" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s887" data-index="8">Centrifuge column at 12000rpm for 3min to remove residual ethanol.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s887" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s888" data-index="9">Place column in new (storage) centrifuge tube. Add 15-30ul elution buffer onto center of membrane. Allow to stand for 2min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s888" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s889" data-index="10">Centrifuge for 50s at 12000rpm to elute DNA. Store at -20˚C. Find concentration collected with Nanodrop.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s889" data-index="10"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
  
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv965-title" data-index="0">Competent Cell Test Kit (iGEM)</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv965-intro" data-index="0">Cell competency test on E.Coli strains with DNA kit provided by iGEM
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m966" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c967" data-index="0,0">Purified DNA (BBa_J04450 RFP construct with plasmid backbone pSB1C3)  at 0.5pg/ul, 5pg/ul, 10pg/ul, 20pg/ul, 50pg/ul (50ul aliquots)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c968" data-index="0,1">Competent cell aliquots</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c969" data-index="0,2">Agar plates with chloramphenicol</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c970" data-index="0,3">SOC media</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s971" data-index="0">Transformation</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s971" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s972" data-index="1">Spin down DNA tubes from Competent Cell Test Kit at 8,000-10,000rpm for 20-30 seconds</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s972" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s973" data-index="2">Thaw competent cells on ice for 10 minutes. Label one 2.0ml microcentrifuge tube for each concentration and pre-chill by placing empty tubes on ice.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s973" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s974" data-index="3">Pipet 1uL of DNA into each microcentrifuge tube. Use a separate tube for each concentration.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s974" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s975" data-index="4">Pipet 50uL of competent cells into each tube, flicking tube gently to mix. Incubate on ice for 30 minutes. Preheat waterbath to 42˚C.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s975" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s976" data-index="5">Heat-shock cells by placing into waterbath for 1 minute.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s976" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s977" data-index="6">Immediately transfer tubes back to ice and incubate on ice for 5 minutes.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s977" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s978" data-index="7">Add 200uL of SOC per tube and incubate at 37˚C for 2 hours. Prepare and label agar plates.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s978" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s979" data-index="8">Pipet 20uL from each tube onto appropriate plate and evenly spread mixture across the plate. Do triplicates of each tube if possible to calculate an average colony yield.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s979" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s980" data-index="9">Incubate at 37˚C overnight or for 16 hours.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s980" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s981" data-index="10">Count number of colonies against a dark background, using an average cell colony count if done in triplicates.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s981" data-index="10"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s982" data-index="11">To measure competent cell efficiency: (colonies on plate) / ng of DNA plated x 1000ng/ug</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s983" data-index="12">To measure ng of DNA plated: 1uL x concentration of DNA in original vial x (volume plated / total reaction volume)</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="11"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s984" data-index="13">Competent cells have an efficiency of 1.5x10^8 to 6x10^8 cfu/ug DNA (cfu = colony-forming unit)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s984" data-index="13"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
  
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<title>Cryostock Glycerol -80°C · Benchling</title>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv717-title" data-index="0">Cryostock Glycerol -80°C</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv717-intro" data-index="0"><span style="color:#000000"><span style="background-color:#FFFFFF">Clonal b</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">acterial </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">freezer </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">stocks are important for preserving </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">plasmid</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">constructs (adapted from Stanford BioE 44 F2015). </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">Make a </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">glycerol </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">freezer </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">stock from your </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">bacterial</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">culture</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">as follows: </span></span>
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m718" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c719" data-index="0,0">50%/40% glycerol</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c720" data-index="0,1">Overnight culture of cells</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c721" data-index="0,2">-<span style="color:#000000"><span style="background-color:#FFFFFF">80°C Freezer  </span></span></div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s722" data-index="0">Making a clonal glycerol freezer stock</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s722" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s723" data-index="1"><span style="color:#000000"><span style="background-color:#FFFFFF">Write down in your no</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">tebook the </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">identifier and the plasmid strain you plan on</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">storing</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">in the tube.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s723" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s724" data-index="2"><span style="color:#000000"><span style="background-color:#FFFFFF">Transfer</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">0.5</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">mL of 50% (or 40%) glycerol</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">into each tube</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s724" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s725" data-index="3"><span style="color:#000000"><span style="background-color:#FFFFFF">Briefly v</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">ortex your </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">bacterial culture </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">to </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">ensure </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">that it is</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">well mixed</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">. Add 0.5</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">mL</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">of the </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">mixed</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">culture to the appropriate</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">ly labeled</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">tube containing the glycerol</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s725" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s726" data-index="4"><span style="color:#000000"><span style="background-color:#FFFFFF">Tightly cap the tube. Gently vortex the tube or invert multiple times to mix.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s726" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s727" data-index="5"><span style="color:#000000"><span style="background-color:#FFFFFF">Access the computer with the 2D barcode scanning hardware </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">attached.</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">Ope</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">n the “BIOE44 </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">F</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">201</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">4</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">glycerol stock” Excel file in the BIOE44 folder on</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">the computer.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s727" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s728" data-index="6"><span style="color:#000000"><span style="background-color:#FFFFFF">Record your unique</span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">identifier into the tube/notebook/spreadshseet </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">and input the appropriate plasmid ID (e.g. pSB1C3, </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">p70417</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">, pComet, </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">pX330</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">-</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">gRNA, </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">or</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">pJ1201</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">-</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">03c, etc.) into the plasmid ID column. </span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">Add into the notes column any</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">not</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">es you deem appropriate. Rememb</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">er to save the file when y</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">ou are </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">finished</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">!</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s728" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s729" data-index="7"><span style="color:#000000"><span style="background-color:#FFFFFF">Store the glycerol stock in a labeled box in the </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">-</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">80°C freezer.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s729" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
  
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<title>Cryostock glycerol revival · Benchling</title>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv709-title" data-index="0">Cryostock glycerol revival</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv709-intro" data-index="0">Revive your frozen culture stocks
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m710" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c711" data-index="0,0">Cryostock</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c712" data-index="0,1">Pipette</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s713" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s713" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s714" data-index="1">Use a pipette tip to scrape off a little bit of the semi-thawed glycerol. Put tip with cells into media
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</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s714" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s715" data-index="2">Put your glycerol stock back into the box in the -80°C freezer. Do not let it thaw completely! Thawing and freezing your stock repeatedly will kill your cells.
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</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s715" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s716" data-index="3">Put inoculated culture into shaking incubator to let culture grow.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s716" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
  
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<title>Capture SELEX · Benchling</title>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv435-title" data-index="0">Capture SELEX</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv435-intro" data-index="0">Written by Michael Becich, Julia Gross, Amy Weissenbach
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Adapted from Lan et al. 2012  (https://www.ideals.illinois.edu/bitstream/handle/2142/42441/Tian_Lan.pdf?sequence=1, http://www.hindawi.com/journals/jamc/2012/415697/)
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m436" data-index="0">Streptavidin Beads </div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c437" data-index="0,0">10mg/mL concentration (2 mL, 20mg total, 0.7-1.2X10^9 beads per mg)</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m438" data-index="1">Random Library</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c439" data-index="1,0">ATA CCA GCT TAT TCA ATT NNN NNN NNN NTG AGG CTC GAT CNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNA GAT AGT AAG TGC AAT CT</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m440" data-index="2">Biotinylated Capture Oligo's (25 nmole)</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c441" data-index="2,0">/5Biosg/TA CCG CAA AAA AAA AGA TCG AGC CTC A</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c442" data-index="2,1">Use 750 pmol oligo for 150uL (1.5mg) beads, as rounds go on should consider cutting down</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m443" data-index="3">PCR Primers (Forward, Reverse, and Elongated Reverse) (25 nmole)</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c444" data-index="3,0">FWD: ATACCAGCTTATTCAATT</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c445" data-index="3,1">RVS: GATTGCACTTACTATCT</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c446" data-index="3,2">RVS_plus_spacer:  AAAAAAAAAA<span style="color:#344043"><span style="background-color:#FFFFFF">AAAAAAAAAA</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">AAAAAAAAAA/iSp18/</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">GATTGCACTTACTATCT</span></span></div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m447" data-index="4">Selection Buffer (SB) - make 200 mL</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c448" data-index="4,0"><span style="color:#000000"><span style="background-color:#FFFFFF">100 mM NaCl  - 0.005mol, about 0.3g/50mL</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c449" data-index="4,1"><span style="color:#000000"><span style="background-color:#FFFFFF">20 mM Tris-HCl pH7.6</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c450" data-index="4,2"><span style="color:#000000"><span style="background-color:#FFFFFF">2 mM MgCl</span></span><sub><span style="color:#000000"><span style="background-color:#FFFFFF">2</span></span></sub><span style="color:#000000"><span style="background-color:#FFFFFF">  -- 0.095g/50mL</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c451" data-index="4,3"><span style="color:#000000"><span style="background-color:#FFFFFF">5 mM KCl -- .0186 g/ 50mL</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c452" data-index="4,4"><span style="color:#000000"><span style="background-color:#FFFFFF">1 mM CaCl</span></span><sub><span style="color:#000000"><span style="background-color:#FFFFFF">2 </span></span></sub><span style="color:#000000"><span style="background-color:#FFFFFF"> --- </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">0.005549g / 50mL</span></span></div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m453" data-index="5">Binding &amp; Washing Buffer (B&amp;W) - make 120 mL</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c454" data-index="5,0"><span style="color:#000000"><span style="background-color:#FFFFFF">10 mM Tris-HCl pH7.5</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c455" data-index="5,1"><span style="color:#000000"><span style="background-color:#FFFFFF">1 mM EDTA</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c456" data-index="5,2"><span style="color:#000000"><span style="background-color:#FFFFFF">2 M NaCl</span></span></div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m457" data-index="6">TE Buffer </div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c458" data-index="6,0">10mM Tris-HCl pH 7.4</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c459" data-index="6,1">1 mM EDTA</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m460" data-index="7">TBE Buffer</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c461" data-index="7,0">90 mM Tris-Hcl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c462" data-index="7,1">90 mM boric acid</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c463" data-index="7,2">2 mM EDTA</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m464" data-index="8">Sodium Acetate Soln</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c465" data-index="8,0"><span style="color:#344043"><span style="background-color:#FFFFFF">2mM EDTA, 300 mM sodium acetate, and pH 7.8 (5.2)</span></span></div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m466" data-index="9">PCR Rxn Buffer (don't think we need this, Q5 Master Mix)</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c467" data-index="9,0"><span style="color:#000000"><span style="background-color:#FFFFFF">80 mM Tris-HCl pH9.5 </span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c468" data-index="9,1"><span style="color:#000000"><span style="background-color:#FFFFFF">20 mM (NH</span></span><sub><span style="color:#000000"><span style="background-color:#FFFFFF">4</span></span></sub><span style="color:#000000"><span style="background-color:#FFFFFF">)</span></span><sub><span style="color:#000000"><span style="background-color:#FFFFFF">2</span></span></sub><span style="color:#000000"><span style="background-color:#FFFFFF">SO</span></span><sub><span style="color:#000000"><span style="background-color:#FFFFFF">4</span></span></sub></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c469" data-index="9,2"><span style="color:#000000"><span style="background-color:#FFFFFF">0.02% Tween20</span></span></div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m470" data-index="10">PCR Cleanup Kit</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c471" data-index="10,0">TBD: Need one that will work well when amplifying very small DNA sequences</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s472" data-index="0">1. <span style="color:#999999"><span style="background-color:#FFFFFF">Coupling of Capture Oligos to Streptavidin-Coated Magnetic Beads</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s472" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s473" data-index="1">1000 pmol oligo mixture for 200uL magnetic beads (500pmol/mg)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s473" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s474" data-index="2">(Calculate appropriate volume of beads to capture oligo DNA (*dependent on bead type. <span style="color:#333333"><span style="background-color:#FFFFFF">500 pmol ss-oligonucleotides per mg bead for </span></span><span style="color:#333333"><span style="background-color:#FFFFFF">Dynabeads® MyOne™ Streptavidin C1))</span></span></div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s475" data-index="3"><span style="color:#344043"><span style="background-color:#FFFFFF">In a 1.5mL Eppendorf Tube, add 200<b> </b></span></span><b><span style="color:#222222"><span style="background-color:#FFFFFF">μ</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">L beads</span></span></b><span style="color:#344043"><span style="background-color:#FFFFFF"> (2.0mg), wash 3X w/ </span></span><span style="color:#000000"><span style="background-color:#FFFFFF">500 </span></span><i><span style="color:#000000"><span style="background-color:#FFFFFF">μ</span></span></i><span style="color:#000000"><span style="background-color:#FFFFFF">L <b>B&amp;W</b></span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s475" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s476" data-index="4">Separate beads by placing the tube in a magnet stand.</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s477" data-index="5">Resuspend in <span style="color:#000000"><span style="background-color:#FFFFFF">B&amp;W to a concentration of 2mg per mL (2×10</span></span><sup><span style="color:#000000"><span style="background-color:#FFFFFF">9</span></span></sup><span style="color:#000000"><span style="background-color:#FFFFFF"> beads/mL)</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s477" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s478" data-index="6"><span style="color:#000000"><span style="background-color:#FFFFFF">So 2.0mg in 1000uL</span></span></div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s479" data-index="7"> Add equal volume of oligo (1000pmol) and incubate <span style="color:#000000"><span style="background-color:#FFFFFF">at room temperature for 1 h with gentle rotation</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s479" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s480" data-index="8"><span style="color:#000000"><span style="background-color:#FFFFFF">Separate and wash 3X w/ 500 </span></span><i><span style="color:#000000"><span style="background-color:#FFFFFF">μ</span></span></i><span style="color:#000000"><span style="background-color:#FFFFFF">L B&amp;W</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s480" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s481" data-index="9"><span style="color:#000000"><span style="background-color:#FFFFFF">Wash  3X w/ 500 </span></span><i><span style="color:#000000"><span style="background-color:#FFFFFF">μ</span></span></i><span style="color:#000000"><span style="background-color:#FFFFFF">L SB</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s481" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s482" data-index="10">Nanodrop 3rd fraction to confirm no DNA in flow-through (if DNA, wash and repeat)</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s483" data-index="11">Resuspend in selection buffer <span style="color:#000000"><span style="background-color:#FFFFFF">1×10</span></span><sup><span style="color:#000000"><span style="background-color:#FFFFFF">9</span></span></sup><span style="color:#000000"><span style="background-color:#FFFFFF"> beads/mL (</span></span><span style="color:#000000"><span style="background-color:#FFFFFF">1×10<sup>8</sup></span></span><span style="color:#000000"><span style="background-color:#FFFFFF"> beads/mL for each subsequent round), so 2.0 mg in 2mL</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s483" data-index="11"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s484" data-index="12">Store at 4C.  When use, resuspend completely (i.e. vortex etc.)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s484" data-index="12"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s485" data-index="13">Use 1mL for first round</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s486" data-index="14">100uL for following rounds</div></div></li></ul></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s487" data-index="15">2. Preparation of Target</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s487" data-index="15"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s488" data-index="16">Dilute Target to 1mM in selection buffer</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s488" data-index="16"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s489" data-index="17">Sterile filter (used pore size of 0.22um), aquilot and store at -18 <span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span>C</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s489" data-index="17"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s490" data-index="18"><span style="color:#344043"><span style="background-color:#FFFFFF">3. Thermal equilibration of oligonucleotide pool</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s490" data-index="18"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="11"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s491" data-index="19">DNA library <span style="color:#344043"><span style="background-color:#FFFFFF">(2-3nmol 1st round, all in following rounds) heated to 90 </span></span><span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">C for 8min</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s491" data-index="19"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s492" data-index="20">Immediately cooled and kept at 4 <span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span>C for 10min</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="12"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s493" data-index="21">At same time, wash streptavidin bead-capture oligo complexes (1x10^9 and 1x10^8 in following) around 3X w/ 500uL SB</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s493" data-index="21"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s494" data-index="22">Use Nanodrop to confirm no more DNA washing off</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="13"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s495" data-index="23">Bead-capture oligo complexes resuspended in 300uL of the pretreated DNA library</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s495" data-index="23"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s496" data-index="24">Incubate overnight at 21 <span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span>C w/ mild shaking</div></div></li></ul></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s497" data-index="25">4. Capture-SELEX round</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s497" data-index="25"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="14"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s498" data-index="26">Wash beads around 9X w/ 500uL SB to remove unbound DNA</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s498" data-index="26"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s499" data-index="27">Nanodrop to determine fraction coming off, place this fraction aside for comparison</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="15"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s500" data-index="28">Incubate DNA-bead complexes in 500uL SB at 28 <span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span>C for 15min w/ mild shaking</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s500" data-index="28"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="16"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s501" data-index="29">Wash around 7X w/ 500uL SB  (to remove all remaining unhybridized or weakened DNA structures)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s501" data-index="29"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s502" data-index="30">Nanodrop to determine fraction coming off</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s503" data-index="31">Save flow-through for comparison, label</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="17"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s504" data-index="32">Suspend DNA-bead complexes in 300uL SB</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s504" data-index="32"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s505" data-index="33">Incubate at 21 <span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span>C for 45min w/ mild shaking</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="18"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s506" data-index="34">Wash around 7 X with 500uL SB</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s506" data-index="34"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s507" data-index="35">Nanodrop to measure fraction coming off</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s508" data-index="36">Save flow-through, label</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="19"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s509" data-index="37">Suspend DNA-bead complexes in 300uL target mixture</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s509" data-index="37"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s510" data-index="38">Incubate at 21<span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span>C for 45min w/ mild shaking</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="20"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s511" data-index="39">Use a magnet to separate out the beads, saving the remaining liquid</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s511" data-index="39"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="21"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s512" data-index="40">Save 10% of the flow-through as stock solution (labeling as necessary), use rest for PCR amplification in the following step.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s512" data-index="40"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s513" data-index="41">5. PCR Step</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s513" data-index="41"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="22"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s514" data-index="42">Nanodrop, determine appropriate number of parallel PCR reactions (e.g. 15, 100uL each)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s514" data-index="42"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s515" data-index="43">FOR Q5 High Fidelity Master Mix, 50uL rxn:</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s516" data-index="44">Q5 Master Mix: 25uL</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s517" data-index="45">10uM forward primer: 2.5uL</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s518" data-index="46">10uM reverse primer: 2.5uL</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s519" data-index="47">DNA, properly diluted: 20uL</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="23"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s520" data-index="48">For Q5 Master Mix, set thermocycler to </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s520" data-index="48"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s521" data-index="49">98<span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">C for 30s</span></span></div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s522" data-index="50">(98<span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">C for 5s</span></span></div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s523" data-index="51">51<span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">C for 10s</span></span></div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s524" data-index="52">72<span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">C for 20s) repeat X times</span></span></div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s525" data-index="53">72<span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">C for 2min</span></span></div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s526" data-index="54">4-10<span style="color:#344043"><span style="background-color:#FFFFFF">°</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">C for </span></span>∞</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="24"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s527" data-index="55">&lt;Gel electrophoresis on 2.5% agarose gel to confirm proper length of DNA product (if feel compelled to do so)&gt;</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s527" data-index="55"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="25"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s528" data-index="56">Pool PCR product and precipitate with ethanol in presence of linear polyacrylamide [19]</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s528" data-index="56"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s529" data-index="57">Add ethanol 2.5-3-fold to solution</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s530" data-index="58">Linear polyacrylamide ($30/tube) 10-20 ug/mL</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s531" data-index="59">Centrifuge for 45min (4-10C)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s532" data-index="60">Pellet will be on outside edge (always have one side, eg hinge facing out, so know where pellet it)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s533" data-index="61">Pour most supernatant off, then pipet rest (could save in case don't end up with anything)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s534" data-index="62">Use kimwipe to touch top of tube</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s535" data-index="63">If still stuff left, use micropipettor to get last bit out</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s536" data-index="64">leave open so that rest can evaporate, <span style="color:#344043"><span style="background-color:#FFFFFF">til looks dry</span></span></div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s537" data-index="65">(COULD put in vaccum centrifuge, but be careful bc DNA can get lodged to side)</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="26"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s538" data-index="66">Resuspend product in 100uL TE buffer</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s538" data-index="66"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="27"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s539" data-index="67">Run denaturing PAGE in TBE buffer</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s539" data-index="67"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s540" data-index="68">-Run with two extra lanes dedicated to 1) a DNA ladder and 2) a PCR product sample that you will later stain as a reference (making the sample unusable) </div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s541" data-index="69">-When finish running, slice out the lanes you intend to stain.  Follow gel-staining protocol. (We used Gel Red.)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s542" data-index="70">-Line the stained gel next to the unstained gel in a UV box.  First, identify the <span style="color:#344043"><span style="background-color:#FFFFFF">confirm the presence of the correct DNA bands in the stained PCR product lane.  There should be one 100bp fragment and one slightly longer fragment. Next, u</span></span>se the stained gel as a reference to cut out the unstained (and therefore invisible) 100bp fragments in the adjacent lanes.  Use these unstained gel pieces in the following step.</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="28"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s543" data-index="71">Elute DNA from gel with 2mM EDTA, 300 mM sodium acetate, and pH 7.8 (5.2)(around 5mL of this solution) -- adding acetic acid</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s543" data-index="71"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s544" data-index="72">Set at 80C for 150min with mild shaking</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="29"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s545" data-index="73"> Precipitate DNA with ethanol in presence of linear polyacrylamide [19]</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s545" data-index="73"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s546" data-index="74">Break into 1.7mL tubes</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s547" data-index="75">Add ethanol 2.5-3-fold sodium acetate solution</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s548" data-index="76">Linear polyacrylamide ($30/tube) 10-20 ug/mL</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s549" data-index="77">Centrifuge for 45min (4-10C)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s550" data-index="78">Pellet will be on outside edge (always have one side, eg hinge facing out, so know where pellet it)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s551" data-index="79">Pour most supernatant off, then pipet rest (could save in case don't end up with anything)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s552" data-index="80">use kimwipe to touch top of tube</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s553" data-index="81">if still stuff left, use micropipettor to get last bit out</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s554" data-index="82">leave open so that rest can evaporate, <span style="color:#344043"><span style="background-color:#FFFFFF">til looks dry</span></span></div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s555" data-index="83">COULD put in vaccum centrifuge, but be careful bc DNA can get lodged to side</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="30"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s556" data-index="84">Resuspend DNA in SB.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s556" data-index="84"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s557" data-index="85">7. TEST - TBD</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s557" data-index="85"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="31"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s558" data-index="86">&lt;goal of around 13 rounds&gt;</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s558" data-index="86"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="32"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s559" data-index="87">cloning+transformation into chemically competent cells</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s559" data-index="87"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="33"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s560" data-index="88">+sequencing -- talk to Mark</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s560" data-index="88"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s561" data-index="89">could do rounds 7,8</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s562" data-index="90">high throughput sequencing</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="34"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s563" data-index="91">Discuss SPR with Michaela</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s563" data-index="91"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="35"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s564" data-index="92">Archive roughly 10% of each round and save as stock solution</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s564" data-index="92"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
  
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv769-title" data-index="0">Distribution Kit Hydration</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv769-intro" data-index="0">Get started by giving your protocol a name and editing this introduction.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m770" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c771" data-index="0,0">milliQ Water</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s772" data-index="0">To use the DNA in the Distribution Kit, follow these instructions:</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s772" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s773" data-index="1"><i>Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/ul</i></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s773" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s774" data-index="2">With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s774" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s775" data-index="3">Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be <b>red</b>, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s775" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s776" data-index="4"><a href="http://parts.igem.org/Help:Transformation_Protocol" rel="nofollow" target="_blank">Transform</a> 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s776" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s777" data-index="5">Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s777" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s778" data-index="6">Use the resulting culture to <a href="http://openwetware.org/wiki/Miniprep/Kit-free_high-throughput_protocol" rel="nofollow" target="_blank">miniprep</a> the DNA AND make your own glycerol stock (for further instruction on making a glycerol see <a href="http://openwetware.org/wiki/Endy:Making_a_long_term_stock_of_bacteria" rel="nofollow" target="_blank">this page</a>). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s778" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s779" data-index="7"><i>* To know which antibiotics to use, look at the plasmid that the part is in. The </i><a href="http://parts.igem.org/wiki/index.php/Help:Plasmids/Nomenclature" rel="nofollow" target="_blank"><i>naming scheme</i></a><i> for plasmids is specifically designed to indicate antibiotic resistance.</i></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s779" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv833-title" data-index="0">DNA Miniprep</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv833-intro" data-index="0">Protocol for miniprep comes from Epoch kit in lab.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m834" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c835" data-index="0,0">Epoch miniprep kit</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s836" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s836" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s837" data-index="1">Rinse 500ml cylinder with water then ethanol.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s837" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s838" data-index="2">Measure 120ml ethanol, add to wN buffer.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s838" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s839" data-index="3">Add 180ml ethanol to WS buffer.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s839" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s840" data-index="4">Centrifuge liquid culture tubes at 4500rpm (max) for 2min. Be sure to balance tubes.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s840" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s841" data-index="5">Resuspend cell pellet in 200ul MX1 buffer kept in fridge.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s841" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s842" data-index="6">Add 250ul MX2 buffer and gently mix. Incubate at room temp for 1-5 min, don't vortex.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s842" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s843" data-index="7">Add 350ul MX3 and gently mix immediately.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s843" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s844" data-index="8">Centrifuge for 5-10 min at 14,500rpm.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s844" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s845" data-index="9">Transfer supernatant carefully to column.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s845" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s846" data-index="10">Centrifuge 30-60s at 5000rpm. Discard flow-through.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s846" data-index="10"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="11"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s847" data-index="11">Wash column once with 500ul WN buffer by centrifuging for 30s at 9000rpm. Discard flow-through.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s847" data-index="11"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="12"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s848" data-index="12">Wash column once with 700ul WS buffer by centrifuging for 30s at 9000rpm. Discard flow-through.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s848" data-index="12"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="13"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s849" data-index="13">Centrifuge column at 13000rpm for 2min to remove ethanol residue.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s849" data-index="13"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="14"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s850" data-index="14">Place column into new 1.5ml centrifuge tube, add 50ul Elution Buffer onto center of membrane.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s850" data-index="14"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="15"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s851" data-index="15">Stand for 2min at room temp, then centrifuge for 30s at 13000rpm to elute DNA.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s851" data-index="15"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="16"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s852" data-index="16">Store plasmid DNA at 4˚C or -20˚C.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s852" data-index="16"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Electrophoresis · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv942-title" data-index="0">Electrophoresis</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv942-intro" data-index="0">Standard gel electrophoresis.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m943" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c944" data-index="0,0">DNA</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c945" data-index="0,1">DI water</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c946" data-index="0,2">Loading dye</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s947" data-index="0">Electrophoresis</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s947" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s948" data-index="1">Load 1ul molecular ladder into first row.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s948" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s949" data-index="2">Mix 1.5ul purple loading dye, 1ul DNA, 7.5ul DI water on a sheet of parafilm.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s949" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s950" data-index="3">Load each row you're going to run.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s950" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s951" data-index="4">Plug in cables and switch on machine to +/- 100V for +/- 40min. Too high voltage will decrease resolution of results but will run more quickly.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s951" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s952" data-index="5">Ensure it doesn't run off the end.</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s953" data-index="6">Ensure cables are the right way around (black - near DNA, red + far from DNA)</div></div></li></ul></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Gel recipe · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv807-title" data-index="0">Gel recipe</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv807-intro" data-index="0">Making gels
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m808" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c809" data-index="0,0">TAE</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c810" data-index="0,1">Agarose powder</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c811" data-index="0,2">Box molds</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s812" data-index="0">Gel</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s812" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s813" data-index="1">Mix TAE and agarose (~1g powder per 200ml buffer (slightly less for long DNA)).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s813" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s814" data-index="2">Microwave and mix until completely dissolved, pause microwaving when it bubbles.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s814" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s815" data-index="3">Cool until can touch it. Add gel red and mix.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s815" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s816" data-index="4">Carefully pour into box mold and cool at room temperature, covered with plastic wrap. Insert comb.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s816" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s817" data-index="5">Before running gel, ensure TAE in box is just above gel (+/- 400ml).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s817" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Gibson Assembly · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv565-title" data-index="0">Gibson Assembly</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv565-intro" data-index="0">Get started by giving your protocol a name and editing this introduction.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m566" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c567" data-index="0,0">Gibson Master Mix 2x (exonuclase, polymerase, ligase)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c568" data-index="0,1">His tags (annealed)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c569" data-index="0,2">Digested vectors</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c570" data-index="0,3">H2O</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c571" data-index="0,4">LB</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c572" data-index="0,5">Agar</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s573" data-index="0">Gibson ratios</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s573" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s574" data-index="1">Gibson Master Mix 2x: 5ul x 3 = 15ul</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s574" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s575" data-index="2">His tags: 1ul x 3 = 3ul</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s575" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s576" data-index="3">Digested vectors: 1ul</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s576" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s577" data-index="4">H2O: 3ul x 3 = 9ul</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s577" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s578" data-index="5">25g/L LB, 15g/L agar</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s578" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s579" data-index="6">Gibson Times</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s579" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s580" data-index="7">Optional: 48˚C for 5min, 5 @ 45</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s580" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s581" data-index="8">50˚C for 15min, 10 @ 50</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s581" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s582" data-index="9">Optional: 52˚C for 5min, 5 @ 55</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s582" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s583" data-index="10">4˚C forever</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s583" data-index="10"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<title>Gibson Assembly on Geneious · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv922-title" data-index="0">Gibson Assembly on Geneious</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv922-intro" data-index="0">Make plasmids on the internet!
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m923" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c924" data-index="0,0"></div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s925" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s925" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s926" data-index="1">Linearise vector plasmid where you want to insert new gene (eg using a restriction site).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s926" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s927" data-index="2">Manually replace start codon if lost.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s927" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s928" data-index="3">Select the gene you want to insert excl. stop codon and extract.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s928" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s929" data-index="4">Select vector and extracted gene and click tools --&gt; cloning --&gt; Gibson assembly.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s929" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s930" data-index="5">Ensure backbone = vector, box contains gene fragment. Choose min overlap length (18-22), min overlap Tm (48), calculation of Tm is based on protocol.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s930" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s931" data-index="6">* Can also insert multiple genes simultaneously or a subfolder (eg promoter) to make several different products.</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s932" data-index="7">* We want G-C at primer ends, particularly on 5' side.</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s933" data-index="8">* Make sure primer code is written 5'-3' from L--&gt;R for synthesis.</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s934" data-index="9">* If % GC is high then higher annealing T, lower % GC has lower annealing T. High Tm means less likely to anneal in funny places but don't want too high that doesn't get a chance to anneal before enzyme runs at ~72˚C. </div></div></li></ul></div></div></div></div></div></div></div></div></div></div></div>
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<meta charset="utf-8"/>
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<title>Gluconobacter media · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv853-title" data-index="0">Gluconobacter media</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv853-intro" data-index="0">This might optimize cellulose production.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m854" data-index="0">Yeast extract (5g/L)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m855" data-index="1">Peptone (3g/L)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m856" data-index="2">Mannitol (25g/L)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m857" data-index="3">DI water</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m858" data-index="4">HCl (to taste)</div><ul class="components"></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s859" data-index="0">After performing the necessary calculations, weigh out an appropriate amount of each component and fill with water. Adjust the pH to about 5.5, make sure the liquid is in a large enough container to prevent any boiling over, and autoclave.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s859" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div></div></div></div></div></div></div></div></div></div></div>
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<meta charset="utf-8"/>
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<title>Golden Gate Procedure without Master Mix · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv589-title" data-index="0">Golden Gate Procedure without Master Mix</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv589-intro" data-index="0">This may not be as optimized as the NEB formulation, but it has worked before. Good for removing a fragment via restriction digest with a Type IIS Restriction Enzyme or an actual Golden Gate assembly (but try not to use SapI for real Golden Gate because it only has a 3bp overhang).
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m590" data-index="0">The DNA You Need (It's not a bad idea to linearize plasmids)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m591" data-index="1">DI H<sub>2</sub>O</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m592" data-index="2">PCR Tube</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m593" data-index="3">Thermal Cycler</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m594" data-index="4">Everything Important but DNA (It's a good master mix candidate)</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c595" data-index="4,0">5 μL T4 DNA Ligase</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c596" data-index="4,1">2 μL T4 DNA Ligase Buffer</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c597" data-index="4,2">1 μL Type IIS Restriction Enzyme (we have SapI and BsaI)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c598" data-index="4,3">0.2 μL 100X or 1 μL 20X BSA (Bovine Serum Albumin)</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s599" data-index="0">Everything</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s599" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s600" data-index="1">Add the non-DNA components to a new PCR tube</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s600" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s601" data-index="2">Add an appropriate amount of DNA (75 ng per component is recommended)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s601" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s602" data-index="3">Fill to 20 μL with DI H<sub>2</sub>O</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s602" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s603" data-index="4">For 1-4 fragments, incubate at 37°C for 1 hr and 55°C for 5 min. For 5-10 fragments, alternate between <span style="color:#344043"><span style="background-color:#FFFFFF">37°C and 16</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">°C for one minute each for 30 cycles, then  incubate for 5 min at 55</span></span><span style="color:#344043"><span style="background-color:#FFFFFF">°C. For more than 10 fragments, consider going that extra mile and buying the optimized master mix.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s603" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s604" data-index="5">Proceed directly to transfomation.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s604" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<meta charset="utf-8"/>
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<title>H2O2/Peroxidase (Amplex Red) Assay · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv730-title" data-index="0">H2O2/Peroxidase (Amplex Red) Assay</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv730-intro" data-index="0">Used to detect presence of hydrogen peroxide (H2O2) or peroxidase activity in biological samples. Specifically used to detect the activity of lysyl oxidase in coupling monomers.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m731" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c732" data-index="0,0">H2O2/Peroxidase/Amplex Red Assay kit from Thermofisher</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c733" data-index="0,1">96 well plate</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c734" data-index="0,2">plate reader</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s735" data-index="0">Prep for stock solutions</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s735" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s736" data-index="1">Let all reagents in the kit to completely warm to RT before opening.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s736" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s737" data-index="2"><b>10 mM Amplex Red reagent stock soln: </b><span style="color:#E14516">It is <b>AIR SENSITIVE</b>, use the reagent on the same day. Also <b>light sensitive</b>. Use on the same day it's prepared.</span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s737" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s738" data-index="3">Let Amplex Red reagent (Component A, blue cap) and DMSO (Component B, green cap) warm to room temp.<b> Just before using,</b> dissolve Amplex Red reagent into 60 uL of DMSO.</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s739" data-index="4"><b>1X Rxn buffer: </b>Add 4 mL of 5x Reaction buffer (Component C, white cap) to 16 mL of dH2O.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s739" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s740" data-index="5"><b>10 U/mL Horseradish peroxidase stock soln: </b>Dissolve HRP (Component D, yellow cap) in 1.0 mL of 1x Rxn buffer.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s740" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s741" data-index="6">Divide any unused HRP stock soln into single use aliquots and store frozen at -20C.</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s742" data-index="7"><b>20 mM H2O2 working soln:</b> Dilute ~3% H2O2 (Component E, red cap) into appropriate vol of 1X Rxn buffer to make 20 mM H2O2. Actual concentration of H2O2 is indicated on the label.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s742" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s743" data-index="8"><span style="color:#E14516">20 mM working solution eeds to be used within a few hours of prep</span></div></div></li></ul></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s744" data-index="9">H2O2 Assay</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s744" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s745" data-index="10"><b>Prepare H2O2 standard curve</b>. Dilute H2O2 containing samples in 1X Rxn buffer to produce H2O2 concentrations of 0-10 uM.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s745" data-index="10"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s746" data-index="11">Use volume of 50 uL for each rxn. Pipet into individual wells of microplate.</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s747" data-index="12"><b>Prep working soln of 100 uM Amplex Red Reagent/HRP. </b>Mix 50 uL of 10 mM Amplex Red reagent stock soln + 100 uL of 10 U/mL HRP stock soln, 4.85 mL of 1X Rxn buffer.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s747" data-index="12"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s748" data-index="13">Begin rxn. Add50 uL of Amplex Red reagent/HRP working soln to each microplate well containing standards/controls/samples.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s748" data-index="13"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s749" data-index="14">Incubate rxns at room temp for 30 mins, protected from light. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s749" data-index="14"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s750" data-index="15">Use plate reader to detect fluorescence (excitation 530-560 nm, emission 590nm, absorbance 560nm).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s750" data-index="15"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="11"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s751" data-index="16">For each point subtract value derived from no-H2O2 control</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s751" data-index="16"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<meta charset="utf-8"/>
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<title>Inclusion Body Solubilization Reagent · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv798-title" data-index="0">Inclusion Body Solubilization Reagent</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv798-intro" data-index="0">Recover insoluble proteins expressed in inclusion bodies (protein aggregates)
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m799" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c800" data-index="0,0">B-PER Bacterial Protein Extraction Reagent</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c801" data-index="0,1">Inclusion Body Solubilization Reagent</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s802" data-index="0">Protein solubilization</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s802" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s803" data-index="1">Estimate amount of inclusion body prep by subtracting weight of centifuge tube from total weight. Use 8 mL of Inclusion Body Solubilization Reagent per 1 g of wet inclusion body pellet.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s803" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s804" data-index="2">Suspend pellet in appropriate amount of Inclusion Body Solubilization by vortexing or pipetting. Shake suspension of 30 min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s804" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s805" data-index="3">Remove cell debris by centrifugation at 27k x g (15k rpm) for 15 min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s805" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s806" data-index="4">Collect supernatant which contains the solubilized protein. For protein assay, use Coomassie Plus (Bradford) Protein Assay Kit. For SDS-PAGE analysis, remove denaturant by dialysis.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s806" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<meta charset="utf-8"/>
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<title>Large Scale Extraction · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv818-title" data-index="0">Large Scale Extraction</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv818-intro" data-index="0">Get started by giving your protocol a name and editing this introduction.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m819" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c820" data-index="0,0">1 g of wet cell paste or 250 mL bacterial culture with OD600 of ~2.0</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c821" data-index="0,1">CelLytic B</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s822" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s822" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s823" data-index="1">Collect bacterial cells that express protein of interest by centrifuging at 5k x g for 10 min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s823" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s824" data-index="2">Carefully remove the spent medium from the cell pellet. The cell pellet may be frozen or used fresh. Frozen cell pellet will give slightly higher yield of protein</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s824" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s825" data-index="3">Add CelLytic B at ratio of 10-20 mL per gram of cell paste. Mix well to completely resuspend the cells.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s825" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s826" data-index="4">Add lysozyme (final concentration = 0.2 mg/mL) to enhance cell lysis</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s827" data-index="5">Benzonase (final amount of 50 units/mL) to decrease viscosity of solution.</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s828" data-index="6">Protease inhibitors to prevent proteolytic degradation</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s829" data-index="7">Incubate extraction suspension with vortexing/shaking at room temperature for 10-15 minutes to fully extract soluble proteins from cells</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s829" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s830" data-index="8">After cell extraction, centrifuge the extract at 16k x g for 10 mins to pellet insoluble material.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s830" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s831" data-index="9">Carefully remove supernatant containing soluble protein fraction. Another round of extraction will yield more soluble protein if required - but will result in more dilute soluble protein sample</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s831" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s832" data-index="10">Analyze supernatant and insoluble fraction by SDS-PAGE and/or Western  blot to determine which fraction contains protein of interest. For SDS-PAGE use 5-15 uL of each sample for gel.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s832" data-index="10"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<title>Ligating Complementary Strands (Gibson) · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv860-title" data-index="0">Ligating Complementary Strands (Gibson)</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv860-intro" data-index="0">Ligating complementary strands before Gibson Assembly and transformation.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m861" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c862" data-index="0,0">5ul T4 DNA ligase buffer (in freezer in 348 with other restriction enzymes)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c863" data-index="0,1">43ul DI water</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c864" data-index="0,2">1ul forward strands (undiluted)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c865" data-index="0,3">1ul reverse strands (undiluted)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c866" data-index="0,4">2.5ul 1M NaCl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c867" data-index="0,5">PCR tubes</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s868" data-index="0">Ligation</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s868" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s869" data-index="1">Thaw ligase buffer in hand and vortex, then put immediately on ice.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s869" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s870" data-index="2">Mix materials in PCR tubes with a total volume of 52.5ul</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s870" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s871" data-index="3">Set at 95˚C and cool at 0.1˚C on Jessica's thermocycler for 19 minutes</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s871" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s872" data-index="4">Dilute 1:10 if doing Gibson</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s872" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s873" data-index="5">Do Gibson protocol</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s873" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
 +
 
 +
<html lang="en">
 +
<head>
 +
<meta charset="utf-8"/>
 +
<title>Liquid Culture · Benchling</title>
 +
<link href="../../../benchling.css" rel="stylesheet"/>
 +
</head>
 +
<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv912-title" data-index="0">Liquid Culture</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv912-intro" data-index="0">Liquid culture of cells
 +
</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m913" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c914" data-index="0,0">Antibiotic</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c915" data-index="0,1">LB broth</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c916" data-index="0,2">Cell culture</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s917" data-index="0">Liquid Culture</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s917" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s918" data-index="1">Add 5ml LB broth to 15ml tube.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s918" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s919" data-index="2">Add 5ul antibiotic.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s919" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s920" data-index="3">Add +/- 1ul/scrape of cells.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s920" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s921" data-index="4">Shake up and incubate at 37˚C at 180rpm.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s921" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
 +
 
 +
<html lang="en">
 +
<head>
 +
<meta charset="utf-8"/>
 +
<title>Lumio Green Detection · Benchling</title>
 +
<link href="../../../benchling.css" rel="stylesheet"/>
 +
</head>
 +
<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv793-title" data-index="0">Lumio Green Detection</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv793-intro" data-index="0">It's pretty great. You can do this even before inducing.
 +
</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m794" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c795" data-index="0,0"></div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s796" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s796" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s797" data-index="1"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s797" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
 +
 
 +
<html lang="en">
 +
<head>
 +
<meta charset="utf-8"/>
 +
<title>Making LB Agar Plates · Benchling</title>
 +
<link href="../../../benchling.css" rel="stylesheet"/>
 +
</head>
 +
<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv954-title" data-index="0">Making LB Agar Plates</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv954-intro" data-index="0">Making LB agar plates.
 +
</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m955" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c956" data-index="0,0">Agar powder</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c957" data-index="0,1">LB broth</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c958" data-index="0,2">1ml antibiotic/L of liquid</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s959" data-index="0">Making Broth</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s959" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s960" data-index="1">Add agar powder (15g/L) to LB broth.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s960" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s961" data-index="2">Autoclave.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s961" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s962" data-index="3">Cool to 55˚C then add antibiotic. Pour into petri dishes.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s962" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s963" data-index="4">Allow to harden and store inverted at 4˚C in the dark (antibiotic is light sensitive).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s963" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s964" data-index="5">If agar solidifies too early, possible to microwave it but it may explode.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s964" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
 +
 
 +
<html lang="en">
 +
<head>
 +
<meta charset="utf-8"/>
 +
<title>Nanodrop Reader · Benchling</title>
 +
<link href="../../../benchling.css" rel="stylesheet"/>
 +
</head>
 +
<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv904-title" data-index="0">Nanodrop Reader</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv904-intro" data-index="0">Reading DNA concentration with a Nanodrop.
 +
</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m905" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c906" data-index="0,0">Tube of DNA</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s907" data-index="0">Reading DNA</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s907" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s908" data-index="1">Read blank of 1ul elution buffer.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s908" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s909" data-index="2">Run each sample, name at Sample ID, hit measure.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s909" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s910" data-index="3">Want 260/280 + 260/280 ~ 2.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s910" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s911" data-index="4">Record concentrations on stored tubes.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s911" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
 +
 
 +
<html lang="en">
 +
<head>
 +
<meta charset="utf-8"/>
 +
<title>Native PAGE · Benchling</title>
 +
<link href="../../../benchling.css" rel="stylesheet"/>
 +
</head>
 +
<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv584-title" data-index="0">Native PAGE</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv584-intro" data-index="0">Actually this is on a card I don't possess, should be in the lab though. I'll look it up at some point soon. Good luck Anna!
 +
</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m585" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c586" data-index="0,0"></div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s587" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s587" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s588" data-index="1"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s588" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
 +
 
 +
<html lang="en">
 +
<head>
 +
<meta charset="utf-8"/>
 +
<title>PCR · Benchling</title>
 +
<link href="../../../benchling.css" rel="stylesheet"/>
 +
</head>
 +
<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv656-title" data-index="0">PCR</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv656-intro" data-index="0">Get started by giving your protocol a name and editing this introduction. *Primer Tm 50˚C target*
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m657" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c658" data-index="0,0">Taq master mix</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c659" data-index="0,1">Forward primer</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c660" data-index="0,2">Reverse primer</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c661" data-index="0,3">DI water</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c662" data-index="0,4">Template DNA</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c663" data-index="0,5">PCR tubes</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s664" data-index="0">Polymerase Chain Reaction</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s664" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s665" data-index="1">In a PCR tube, mix:</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s665" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s666" data-index="2">12.5ul Taq master mix (green)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s667" data-index="3">1ul 10uM forward primer (1:20 dilution may be needed)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s668" data-index="4">1ul 10uM reverse primer (1:20 dilution may be needed)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s669" data-index="5">10.5ul super DI water</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s670" data-index="6">Touch of template DNA</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s671" data-index="7">Maintain all on ice throughout process.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s671" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s672" data-index="8">Gently mix tube.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s672" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s673" data-index="9">Place in thermocycler and cycle at:</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s673" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s674" data-index="10">94˚C (30s)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s675" data-index="11">Circle 30 times: 94˚C (15-30s), 45-68˚C (15-60s), 68˚C (1min/kb)</div></div></li></ul></div><div class="mediocre-item is-description"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s676" data-index="12">68˚C (5min)</div></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s677" data-index="13">Final hold 4-10˚C.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s677" data-index="13"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Phosphorylation and Ligation of Repetitive DNA Building Blocks · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv678-title" data-index="0">Phosphorylation and Ligation of Repetitive DNA Building Blocks</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv678-intro" data-index="0"><span style="color:#344043"><span style="background-color:#FFFFFF">Use this protocol to phosphorylate and repetitive DNA buidling blocks, and ligate them into randomized long repetitive strands.</span></span>
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m679" data-index="0"><span style="color:#344043"><span style="background-color:#FFFFFF">Materials per Tube (Phosphorylation)      </span></span><u><span style="color:#344043"><span style="background-color:#FFFFFF">Volume</span></span></u></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c680" data-index="0,0">DNA building block mixture (80 µM) 5 µl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c681" data-index="0,1">Ligase 10x Buffer 2 <span style="color:#344043"><span style="background-color:#FFFFFF">µl</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c682" data-index="0,2">DI Water 11.5 µl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c683" data-index="0,3">T4 Polynucleotide Kinase (PNK) 1.5 µl</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m684" data-index="1">Total Volume (Phosphorylation) 20 µl</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m685" data-index="2">Materials per Tube (Ligation)</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c686" data-index="2,0">Phosphorylation mix (above) 20 µl</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c687" data-index="2,1">T4 DNA Ligase 1 µl</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m688" data-index="3">Total Volume (Phosphorylation) 21 µl</div><ul class="components"></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s689" data-index="0">Phosphorylation</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s689" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s690" data-index="1">Place all materials on ice to thaw. If needed, thaw Ligase 10x Buffer in hands and vortex until all white precipitate has disolved. Place back on ice for 2 min before adding to rxn mixture.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s690" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s691" data-index="2">Add materials listed above with desired <span style="color:#344043"><span style="background-color:#FFFFFF">80 µM</span></span> DNA building block mix to PCR tube </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s691" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s692" data-index="3">Incubate in thermal cycler at 37°C for 1 hr</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s692" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s693" data-index="4">Ligation</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s693" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s694" data-index="5">Add 1 µl T4 DNA Ligase to each tube -- vortex and spin down to mix if</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s694" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s695" data-index="6">Incubate at 16°C overnight</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s695" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Scanning a Gel · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv935-title" data-index="0">Scanning a Gel</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv935-intro" data-index="0">Scanning gels.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m936" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c937" data-index="0,0">Gel to read</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s938" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s938" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s939" data-index="1">Carefully remove gel from box and place on scanner.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s939" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s940" data-index="2">Select area on scanner.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s940" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s941" data-index="3">Run scanner.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s941" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Sequencing Order · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv622-title" data-index="0">Sequencing Order</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv622-intro" data-index="0">https://www.elimbio.com/
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m623" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c624" data-index="0,0">1 uL primer_F (VF) diluted 1:20</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c625" data-index="0,1">1 1uL primer_R (VR) diluted 1:20</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c626" data-index="0,2">30ng DNA</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c627" data-index="0,3">15 uL total volume water</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s628" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s628" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s629" data-index="1"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s629" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Small/Trial Scale Extraction · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv630-title" data-index="0">Small/Trial Scale Extraction</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv630-intro" data-index="0">Determine the fraction in which the protein of interest will be found
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m631" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c632" data-index="0,0">Bacterial culture OD600 0.5-1.0</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c633" data-index="0,1">CelLytic B</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s634" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s634" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s635" data-index="1">Use 1.5 mL of bacterial culture with OD600 of 0.5-1.0 and centrifuge the cells at full speed/7k for 2 min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s635" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s636" data-index="2">Remove spun medium and resuspend the cell pellet in 0.4 CelLytic B.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s636" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s637" data-index="3"> </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s637" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s638" data-index="4">Carefully remove the soluble protein fraction from the cell debris. Additional extractions may be performed if required, but will make your soluble protein sample more dilute.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s638" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s639" data-index="5">Analyze supernatant and insoluble fraction by SDS-PAGE and/or Western blot to determine which fraction contains the protein of interest.Fir SDS-PAGE, use 5-15 uL of each sample for gel.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s639" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>T7 Transformations · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv890-title" data-index="0">T7 Transformations</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv890-intro" data-index="0">High efficiency T7 protocol.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m891" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c892" data-index="0,0">See general transformation materials</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s893" data-index="0">Transformations</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s893" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s894" data-index="1">Thaw cells on ice for 10min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s894" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s895" data-index="2">Add 1-5ul contianing 1pg-100ng plasmid DNA (eg from Gibson assembly).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s895" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s896" data-index="3">Place mixture on ice for 30min. Do not mix.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s896" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s897" data-index="4">Heat shock at 42˚C for 10s exactly.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s897" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s898" data-index="5">Place on ice for 5min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s898" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s899" data-index="6">Pipette 950ul room temp SOC.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s899" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s900" data-index="7">Place at 37˚C for 60min, shaking at 250rpm.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s900" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s901" data-index="8">Warm selection plates to 37˚C.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s901" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s902" data-index="9">Mix cells and perform dilutions.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s902" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s903" data-index="10">Spread 50-100ul of cell dilution onto plates. Incubate overnight at 37˚C.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s903" data-index="10"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Transformation: Chemically Competent Cells · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv752-title" data-index="0">Transformation: Chemically Competent Cells</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv752-intro" data-index="0">Transformation protocol for chemically competent cells.
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</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m753" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c754" data-index="0,0">SOC (super optimal broth with catabolite repression)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c755" data-index="0,1">NED 5-alpha competent E.coli cells (Half a stock)</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c756" data-index="0,2">Purified DNA</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c757" data-index="0,3"><span style="color:#344043"><span style="background-color:#FFFFFF">Agar plates with chloramphenicol</span></span></div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s758" data-index="0">Transformation</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s758" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s759" data-index="1">Thaw NEB 5-alpha competent E.coli on ice for 10 minutes (Half a stock is fine, half each subsequent amount)</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s759" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s760" data-index="2">Add 1-5ul of 1pg-100ng plasmid DNA to cell mixture. Flick tube to mix, do not vortex.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s760" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s761" data-index="3">Place on ice for 30min. Do not mix.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s761" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s762" data-index="4">Heat shock at 42˚C for 30s.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s762" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s763" data-index="5">Place on ice for 5min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s763" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s764" data-index="6">Pipette 950ul room temp SOC into mixture (475 if using half stock).</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s764" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s765" data-index="7">Place at 37˚C for 60 minutes, shaking at 250rpm.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s765" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s766" data-index="8">Perform several 10-fold dilutions (1:10, 1:100) of cells.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s766" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s767" data-index="9">Plate 50-100ul of each dilution onto an antibiotic selection plate.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s767" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s768" data-index="10">Incubate overnight at 37˚C.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s768" data-index="10"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
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<html lang="en">
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<head>
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<meta charset="utf-8"/>
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<title>Transformation: Electrocompetent Cells · Benchling</title>
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<link href="../../../benchling.css" rel="stylesheet"/>
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</head>
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<body><div><div class="printable-container"><div><div class="protocolEditor"><div></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv640-title" data-index="0">Transformation: Electrocompetent Cells</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv640-intro" data-index="0">Transformation protocol for electrocompetent cells.
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To make electrocompetent cells, refer to: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939052/.
 +
</div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m641" data-index="0"></div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c642" data-index="0,0"><span style="color:#344043"><span style="background-color:#FFFFFF">Agar plates with chloramphenicol</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c643" data-index="0,1"><span style="color:#344043"><span style="background-color:#FFFFFF">NED 5-alpha competent E.coli cells</span></span></div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c644" data-index="0,2">SOC media</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c645" data-index="0,3">Purified DNA</div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s646" data-index="0">Transformation</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s646" data-index="0"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s647" data-index="1">Place electroporation cuvettes, microcentrifuge, and cuvette holder on ice.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s647" data-index="1"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s648" data-index="2">Thaw NEB 5-alpha electrocompetent E.coli on ice for 10min.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s648" data-index="2"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s649" data-index="3">Transfer 25ul of cells and 1ul DNA into microcentrifuge tube.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s649" data-index="3"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s650" data-index="4">Transfer mixture to cuvette. Make sure all parts are cold to avoid cells exploding.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s650" data-index="4"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s651" data-index="5">Electroporate with the following conditions:</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s651" data-index="5"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s652" data-index="6">Add 975ul SOC in cell box to cuvette, mixing gently, then place in round bottom culture tube.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s652" data-index="6"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s653" data-index="7">Incubate at 37˚C, shaking at 250rpm for 1hr.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s653" data-index="7"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s654" data-index="8">Dilute cells (1:10, 1:100) and spread 100-200ul onto antibiotic selection plate.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s654" data-index="8"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s655" data-index="9">Incubate cells overnight at 37˚C.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s655" data-index="9"></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div></div></div></div></div></div></div></div></div></div></div>
  
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Revision as of 11:43, 19 October 2016


Stanford-Brown 2016

Amide Synthesis with trimethyl borate · Benchling

Amide Synthesis with trimethyl borate

Introduction

Synthesis of amide bonds with trimethyl borate as the coupling reagent. Used to produce polymers from p-aminobenzoic acid monomers.

Materials

    • 2 equiv. P-aminobenzoic acid (pABA)
    • 2 equiv. trimethyl borate
    • Nonpolar/aprotic solvent (MeCN)

Procedure

  • Amide synthesis
  1. Add pABA (1 equiv) to MeCN (2 mL, 0.5 M)
  1. Add 1 equiv trimethyl borate to mixture. Stir at 80C (100C?) for 5-24 hours.
  • Solid Extraction
  1. Dilute reaction mixture with CH2Cl2 or EtOAc (organic solvent) and H2O (polar solvent).
  1. Add MgSO4 (anhydrous) to remove H2O.
Annealing of Template DNA · Benchling

Annealing of Template DNA

Introduction

Use this protocol to anneal complimentary single stranded oligonucleotides.

Materials

  • Measurements per Tube:
    • Forward Seq (200 µM) 10 µl
    • Reverse Seq (200 µM) 10 µl
    • 10x H Buffer 2 µl
    • D.I. Water 28 µl
  • Total Volume per Tube: 50 µl
    • Final Concentration: 80 mM

      Procedure

      • Anneal Strands
      1. Heat mixed tubes at 93°C for 1 min in thermal cycler
      1. Decrease heat of thermal cycler by 5°C every 2 min with small ∆T/sec in between steps
      • When the temp falls more than 20°C below the Tm of the strands to anneal, change steps to decreasing 10°C every min
      1. Once at room temperature, hold samples at 4°C for 15 min, then store samples at -30°C
      BCA Protein Assay · Benchling

      BCA Protein Assay

      Introduction

      For determining concentration of protein using Nanodrop. summarized from http://www.nanodrop.com/Library/T085-NanoDrop-2000-2000c-BCA%20Protocol.pdf

      Materials

      • Working reagent (50A:1B)
        • Pierce BCA Protein Assay Reagent A
        • Pierce BCA Protein Assay Reagent B
      • BSA/Albumin Standard (2mg/mL)
        • PCR tubes/PCR plate

          Procedure

          • Sample Prep
          1. Let all reagents/samples/standards equilibrate to RT.
          1. Prepare enough fresh working reagent (WR) for all standards and samples to be measured using a 50:1 ratio of the kit reagents A:B.
          • If using high-range assay for 5 samples, you'll need approximately 1000 uL of WR total ==> So, you'll need about 1000 uL of A and 20 uL of B (ratio would be 1000:20).
          1. Add reagent volume to each PCR tube.
          • Microassay (1:1 sample to WR) Add 10 uL of WR to each standards and sample tube/well.
          • High range assay (1:20 sample to WR) Add 200 uL of WR to each standards and sample tube/well.
          1. Add 10 uL of standards or samples to the appropriate tube. Mix well by gentle vortexing.
          1. Incubate standard and sample tubes at:
          • 37 C for 30 min OR 60 C for ~5 min. Cool to room temp after.
          Cleaning Electrophoresis Gel · Benchling

          Cleaning Electrophoresis Gel

          Introduction

          Cleaning electrophoresis gel with gel extraction kit (purple).

          Materials

            • 100% ethanol
            • WN + WS buffers

          Procedure

          • Gel Cleaning
          1. Prepare WN + WS buffers by adding appropriate amount of 100% ethanol as shown on bottle.
          1. Carefully cut out piece of gel containing fragment of interest under longwave UV.
          1. Add 300ul GEX for every 100mg gel collected to centrifuge tube and gel piece.
          1. Incubate at 55˚C for 5-10min until gel has dissolved. Can smash with a pipette tip.
          1. Place extraction column into collection tube and load 0.7ul mixture into column. Centrifuge at 5000rpm for 30s. Discard flow-through. Repeat until all mixture is used.
          1. Wash column with 0.5ml (500ul) of WN buffer by centrifuging at 5000rpm for 30s. Discard flow-through.
          1. Wash column with 0.5ml WS buffer by centrifuging for 60s at 5000rpm. Discard flow-through.
          1. Centrifuge column at 12000rpm for 3min to remove residual ethanol.
          1. Place column in new (storage) centrifuge tube. Add 15-30ul elution buffer onto center of membrane. Allow to stand for 2min.
          1. Centrifuge for 50s at 12000rpm to elute DNA. Store at -20˚C. Find concentration collected with Nanodrop.
          Competent Cell Test Kit (iGEM) · Benchling

          Competent Cell Test Kit (iGEM)

          Introduction

          Cell competency test on E.Coli strains with DNA kit provided by iGEM

          Materials

            • Purified DNA (BBa_J04450 RFP construct with plasmid backbone pSB1C3) at 0.5pg/ul, 5pg/ul, 10pg/ul, 20pg/ul, 50pg/ul (50ul aliquots)
            • Competent cell aliquots
            • Agar plates with chloramphenicol
            • SOC media

          Procedure

          • Transformation
          1. Spin down DNA tubes from Competent Cell Test Kit at 8,000-10,000rpm for 20-30 seconds
          1. Thaw competent cells on ice for 10 minutes. Label one 2.0ml microcentrifuge tube for each concentration and pre-chill by placing empty tubes on ice.
          1. Pipet 1uL of DNA into each microcentrifuge tube. Use a separate tube for each concentration.
          1. Pipet 50uL of competent cells into each tube, flicking tube gently to mix. Incubate on ice for 30 minutes. Preheat waterbath to 42˚C.
          1. Heat-shock cells by placing into waterbath for 1 minute.
          1. Immediately transfer tubes back to ice and incubate on ice for 5 minutes.
          1. Add 200uL of SOC per tube and incubate at 37˚C for 2 hours. Prepare and label agar plates.
          1. Pipet 20uL from each tube onto appropriate plate and evenly spread mixture across the plate. Do triplicates of each tube if possible to calculate an average colony yield.
          1. Incubate at 37˚C overnight or for 16 hours.
          1. Count number of colonies against a dark background, using an average cell colony count if done in triplicates.
          • To measure competent cell efficiency: (colonies on plate) / ng of DNA plated x 1000ng/ug
          • To measure ng of DNA plated: 1uL x concentration of DNA in original vial x (volume plated / total reaction volume)
          1. Competent cells have an efficiency of 1.5x10^8 to 6x10^8 cfu/ug DNA (cfu = colony-forming unit)
          Cryostock Glycerol -80°C · Benchling

          Cryostock Glycerol -80°C

          Introduction

          Clonal bacterial freezer stocks are important for preserving plasmid constructs (adapted from Stanford BioE 44 F2015). Make a glycerol freezer stock from your bacterial culture as follows:

          Materials

            • 50%/40% glycerol
            • Overnight culture of cells
            • -80°C Freezer

          Procedure

          • Making a clonal glycerol freezer stock
          1. Write down in your notebook the identifier and the plasmid strain you plan onstoring in the tube.
          1. Transfer 0.5 mL of 50% (or 40%) glycerol into each tube.
          1. Briefly vortex your bacterial culture to ensure that it is well mixed. Add 0.5 mLof the mixed culture to the appropriately labeled tube containing the glycerol
          1. Tightly cap the tube. Gently vortex the tube or invert multiple times to mix.
          1. Access the computer with the 2D barcode scanning hardware attached.Open the “BIOE44 F2014 glycerol stock” Excel file in the BIOE44 folder onthe computer.
          1. Record your unique identifier into the tube/notebook/spreadshseet and input the appropriate plasmid ID (e.g. pSB1C3, p70417, pComet, pX330-gRNA, orpJ1201-03c, etc.) into the plasmid ID column. Add into the notes column anynotes you deem appropriate. Remember to save the file when you are finished!
          1. Store the glycerol stock in a labeled box in the -80°C freezer.
          Cryostock glycerol revival · Benchling

          Cryostock glycerol revival

          Introduction

          Revive your frozen culture stocks

          Materials

            • Cryostock
            • Pipette

          Procedure

          1. Use a pipette tip to scrape off a little bit of the semi-thawed glycerol. Put tip with cells into media
          1. Put your glycerol stock back into the box in the -80°C freezer. Do not let it thaw completely! Thawing and freezing your stock repeatedly will kill your cells.
          1. Put inoculated culture into shaking incubator to let culture grow.
          Capture SELEX · Benchling

          Capture SELEX

          Introduction

          Written by Michael Becich, Julia Gross, Amy Weissenbach Adapted from Lan et al. 2012 (https://www.ideals.illinois.edu/bitstream/handle/2142/42441/Tian_Lan.pdf?sequence=1, http://www.hindawi.com/journals/jamc/2012/415697/)

          Materials

          • Streptavidin Beads
            • 10mg/mL concentration (2 mL, 20mg total, 0.7-1.2X10^9 beads per mg)
          • Random Library
            • ATA CCA GCT TAT TCA ATT NNN NNN NNN NTG AGG CTC GAT CNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNA GAT AGT AAG TGC AAT CT
          • Biotinylated Capture Oligo's (25 nmole)
            • /5Biosg/TA CCG CAA AAA AAA AGA TCG AGC CTC A
            • Use 750 pmol oligo for 150uL (1.5mg) beads, as rounds go on should consider cutting down
          • PCR Primers (Forward, Reverse, and Elongated Reverse) (25 nmole)
            • FWD: ATACCAGCTTATTCAATT
            • RVS: GATTGCACTTACTATCT
            • RVS_plus_spacer: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA/iSp18/GATTGCACTTACTATCT
          • Selection Buffer (SB) - make 200 mL
            • 100 mM NaCl - 0.005mol, about 0.3g/50mL
            • 20 mM Tris-HCl pH7.6
            • 2 mM MgCl2 -- 0.095g/50mL
            • 5 mM KCl -- .0186 g/ 50mL
            • 1 mM CaCl2 --- 0.005549g / 50mL
          • Binding & Washing Buffer (B&W) - make 120 mL
            • 10 mM Tris-HCl pH7.5
            • 1 mM EDTA
            • 2 M NaCl
          • TE Buffer
            • 10mM Tris-HCl pH 7.4
            • 1 mM EDTA
          • TBE Buffer
            • 90 mM Tris-Hcl
            • 90 mM boric acid
            • 2 mM EDTA
          • Sodium Acetate Soln
            • 2mM EDTA, 300 mM sodium acetate, and pH 7.8 (5.2)
          • PCR Rxn Buffer (don't think we need this, Q5 Master Mix)
            • 80 mM Tris-HCl pH9.5
            • 20 mM (NH4)2SO4
            • 0.02% Tween20
          • PCR Cleanup Kit
            • TBD: Need one that will work well when amplifying very small DNA sequences

          Procedure

          • 1. Coupling of Capture Oligos to Streptavidin-Coated Magnetic Beads
          1. 1000 pmol oligo mixture for 200uL magnetic beads (500pmol/mg)
          • (Calculate appropriate volume of beads to capture oligo DNA (*dependent on bead type. 500 pmol ss-oligonucleotides per mg bead for Dynabeads® MyOne™ Streptavidin C1))
          1. In a 1.5mL Eppendorf Tube, add 200 μL beads (2.0mg), wash 3X w/ 500 μL B&W
          • Separate beads by placing the tube in a magnet stand.
          1. Resuspend in B&W to a concentration of 2mg per mL (2×109 beads/mL)
          • So 2.0mg in 1000uL
          1. Add equal volume of oligo (1000pmol) and incubate at room temperature for 1 h with gentle rotation
          1. Separate and wash 3X w/ 500 μL B&W
          1. Wash 3X w/ 500 μL SB
          • Nanodrop 3rd fraction to confirm no DNA in flow-through (if DNA, wash and repeat)
          1. Resuspend in selection buffer 1×109 beads/mL (1×108 beads/mL for each subsequent round), so 2.0 mg in 2mL
          1. Store at 4C. When use, resuspend completely (i.e. vortex etc.)
          • Use 1mL for first round
          • 100uL for following rounds
          • 2. Preparation of Target
          1. Dilute Target to 1mM in selection buffer
          1. Sterile filter (used pore size of 0.22um), aquilot and store at -18 °C
          • 3. Thermal equilibration of oligonucleotide pool
          1. DNA library (2-3nmol 1st round, all in following rounds) heated to 90 °C for 8min
          • Immediately cooled and kept at 4 °C for 10min
          1. At same time, wash streptavidin bead-capture oligo complexes (1x10^9 and 1x10^8 in following) around 3X w/ 500uL SB
          • Use Nanodrop to confirm no more DNA washing off
          1. Bead-capture oligo complexes resuspended in 300uL of the pretreated DNA library
          • Incubate overnight at 21 °C w/ mild shaking
          • 4. Capture-SELEX round
          1. Wash beads around 9X w/ 500uL SB to remove unbound DNA
          • Nanodrop to determine fraction coming off, place this fraction aside for comparison
          1. Incubate DNA-bead complexes in 500uL SB at 28 °C for 15min w/ mild shaking
          1. Wash around 7X w/ 500uL SB (to remove all remaining unhybridized or weakened DNA structures)
          • Nanodrop to determine fraction coming off
          • Save flow-through for comparison, label
          1. Suspend DNA-bead complexes in 300uL SB
          • Incubate at 21 °C for 45min w/ mild shaking
          1. Wash around 7 X with 500uL SB
          • Nanodrop to measure fraction coming off
          • Save flow-through, label
          1. Suspend DNA-bead complexes in 300uL target mixture
          • Incubate at 21°C for 45min w/ mild shaking
          1. Use a magnet to separate out the beads, saving the remaining liquid
          1. Save 10% of the flow-through as stock solution (labeling as necessary), use rest for PCR amplification in the following step.
          • 5. PCR Step
          1. Nanodrop, determine appropriate number of parallel PCR reactions (e.g. 15, 100uL each)
          • FOR Q5 High Fidelity Master Mix, 50uL rxn:
          • Q5 Master Mix: 25uL
          • 10uM forward primer: 2.5uL
          • 10uM reverse primer: 2.5uL
          • DNA, properly diluted: 20uL
          1. For Q5 Master Mix, set thermocycler to
          • 98°C for 30s
          • (98°C for 5s
          • 51°C for 10s
          • 72°C for 20s) repeat X times
          • 72°C for 2min
          • 4-10°C for
          1. <Gel electrophoresis on 2.5% agarose gel to confirm proper length of DNA product (if feel compelled to do so)>
          1. Pool PCR product and precipitate with ethanol in presence of linear polyacrylamide [19]
          • Add ethanol 2.5-3-fold to solution
          • Linear polyacrylamide ($30/tube) 10-20 ug/mL
          • Centrifuge for 45min (4-10C)
          • Pellet will be on outside edge (always have one side, eg hinge facing out, so know where pellet it)
          • Pour most supernatant off, then pipet rest (could save in case don't end up with anything)
          • Use kimwipe to touch top of tube
          • If still stuff left, use micropipettor to get last bit out
          • leave open so that rest can evaporate, til looks dry
          • (COULD put in vaccum centrifuge, but be careful bc DNA can get lodged to side)
          1. Resuspend product in 100uL TE buffer
          1. Run denaturing PAGE in TBE buffer
          • -Run with two extra lanes dedicated to 1) a DNA ladder and 2) a PCR product sample that you will later stain as a reference (making the sample unusable)
          • -When finish running, slice out the lanes you intend to stain. Follow gel-staining protocol. (We used Gel Red.)
          • -Line the stained gel next to the unstained gel in a UV box. First, identify the confirm the presence of the correct DNA bands in the stained PCR product lane. There should be one 100bp fragment and one slightly longer fragment. Next, use the stained gel as a reference to cut out the unstained (and therefore invisible) 100bp fragments in the adjacent lanes. Use these unstained gel pieces in the following step.
          1. Elute DNA from gel with 2mM EDTA, 300 mM sodium acetate, and pH 7.8 (5.2)(around 5mL of this solution) -- adding acetic acid
          • Set at 80C for 150min with mild shaking
          1. Precipitate DNA with ethanol in presence of linear polyacrylamide [19]
          • Break into 1.7mL tubes
          • Add ethanol 2.5-3-fold sodium acetate solution
          • Linear polyacrylamide ($30/tube) 10-20 ug/mL
          • Centrifuge for 45min (4-10C)
          • Pellet will be on outside edge (always have one side, eg hinge facing out, so know where pellet it)
          • Pour most supernatant off, then pipet rest (could save in case don't end up with anything)
          • use kimwipe to touch top of tube
          • if still stuff left, use micropipettor to get last bit out
          • leave open so that rest can evaporate, til looks dry
          • COULD put in vaccum centrifuge, but be careful bc DNA can get lodged to side
          1. Resuspend DNA in SB.
          • 7. TEST - TBD
          1. <goal of around 13 rounds>
          1. cloning+transformation into chemically competent cells
          1. +sequencing -- talk to Mark
          • could do rounds 7,8
          • high throughput sequencing
          1. Discuss SPR with Michaela
          1. Archive roughly 10% of each round and save as stock solution
          Distribution Kit Hydration · Benchling

          Distribution Kit Hydration

          Introduction

          Get started by giving your protocol a name and editing this introduction.

          Materials

            • milliQ Water

          Procedure

          • To use the DNA in the Distribution Kit, follow these instructions:
          1. Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/ul
          1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
          1. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
          1. Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
          1. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
          1. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.
          1. * To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.
          DNA Miniprep · Benchling

          DNA Miniprep

          Introduction

          Protocol for miniprep comes from Epoch kit in lab.

          Materials

            • Epoch miniprep kit

          Procedure

          1. Rinse 500ml cylinder with water then ethanol.
          1. Measure 120ml ethanol, add to wN buffer.
          1. Add 180ml ethanol to WS buffer.
          1. Centrifuge liquid culture tubes at 4500rpm (max) for 2min. Be sure to balance tubes.
          1. Resuspend cell pellet in 200ul MX1 buffer kept in fridge.
          1. Add 250ul MX2 buffer and gently mix. Incubate at room temp for 1-5 min, don't vortex.
          1. Add 350ul MX3 and gently mix immediately.
          1. Centrifuge for 5-10 min at 14,500rpm.
          1. Transfer supernatant carefully to column.
          1. Centrifuge 30-60s at 5000rpm. Discard flow-through.
          1. Wash column once with 500ul WN buffer by centrifuging for 30s at 9000rpm. Discard flow-through.
          1. Wash column once with 700ul WS buffer by centrifuging for 30s at 9000rpm. Discard flow-through.
          1. Centrifuge column at 13000rpm for 2min to remove ethanol residue.
          1. Place column into new 1.5ml centrifuge tube, add 50ul Elution Buffer onto center of membrane.
          1. Stand for 2min at room temp, then centrifuge for 30s at 13000rpm to elute DNA.
          1. Store plasmid DNA at 4˚C or -20˚C.
          Electrophoresis · Benchling

          Electrophoresis

          Introduction

          Standard gel electrophoresis.

          Materials

            • DNA
            • DI water
            • Loading dye

          Procedure

          • Electrophoresis
          1. Load 1ul molecular ladder into first row.
          1. Mix 1.5ul purple loading dye, 1ul DNA, 7.5ul DI water on a sheet of parafilm.
          1. Load each row you're going to run.
          1. Plug in cables and switch on machine to +/- 100V for +/- 40min. Too high voltage will decrease resolution of results but will run more quickly.
          • Ensure it doesn't run off the end.
          • Ensure cables are the right way around (black - near DNA, red + far from DNA)
          Gel recipe · Benchling

          Gel recipe

          Introduction

          Making gels

          Materials

            • TAE
            • Agarose powder
            • Box molds

          Procedure

          • Gel
          1. Mix TAE and agarose (~1g powder per 200ml buffer (slightly less for long DNA)).
          1. Microwave and mix until completely dissolved, pause microwaving when it bubbles.
          1. Cool until can touch it. Add gel red and mix.
          1. Carefully pour into box mold and cool at room temperature, covered with plastic wrap. Insert comb.
          1. Before running gel, ensure TAE in box is just above gel (+/- 400ml).
          Gibson Assembly · Benchling

          Gibson Assembly

          Introduction

          Get started by giving your protocol a name and editing this introduction.

          Materials

            • Gibson Master Mix 2x (exonuclase, polymerase, ligase)
            • His tags (annealed)
            • Digested vectors
            • H2O
            • LB
            • Agar

          Procedure

          • Gibson ratios
          1. Gibson Master Mix 2x: 5ul x 3 = 15ul
          1. His tags: 1ul x 3 = 3ul
          1. Digested vectors: 1ul
          1. H2O: 3ul x 3 = 9ul
          1. 25g/L LB, 15g/L agar
          • Gibson Times
          1. Optional: 48˚C for 5min, 5 @ 45
          1. 50˚C for 15min, 10 @ 50
          1. Optional: 52˚C for 5min, 5 @ 55
          1. 4˚C forever
          Gibson Assembly on Geneious · Benchling

          Gibson Assembly on Geneious

          Introduction

          Make plasmids on the internet!

          Materials

          Procedure

          1. Linearise vector plasmid where you want to insert new gene (eg using a restriction site).
          1. Manually replace start codon if lost.
          1. Select the gene you want to insert excl. stop codon and extract.
          1. Select vector and extracted gene and click tools --> cloning --> Gibson assembly.
          1. Ensure backbone = vector, box contains gene fragment. Choose min overlap length (18-22), min overlap Tm (48), calculation of Tm is based on protocol.
          • * Can also insert multiple genes simultaneously or a subfolder (eg promoter) to make several different products.
          • * We want G-C at primer ends, particularly on 5' side.
          • * Make sure primer code is written 5'-3' from L-->R for synthesis.
          • * If % GC is high then higher annealing T, lower % GC has lower annealing T. High Tm means less likely to anneal in funny places but don't want too high that doesn't get a chance to anneal before enzyme runs at ~72˚C.
          Gluconobacter media · Benchling

          Gluconobacter media

          Introduction

          This might optimize cellulose production.

          Materials

          • Yeast extract (5g/L)
            • Peptone (3g/L)
              • Mannitol (25g/L)
                • DI water
                  • HCl (to taste)

                    Procedure

                    • After performing the necessary calculations, weigh out an appropriate amount of each component and fill with water. Adjust the pH to about 5.5, make sure the liquid is in a large enough container to prevent any boiling over, and autoclave.
                    Golden Gate Procedure without Master Mix · Benchling

                    Golden Gate Procedure without Master Mix

                    Introduction

                    This may not be as optimized as the NEB formulation, but it has worked before. Good for removing a fragment via restriction digest with a Type IIS Restriction Enzyme or an actual Golden Gate assembly (but try not to use SapI for real Golden Gate because it only has a 3bp overhang).

                    Materials

                    • The DNA You Need (It's not a bad idea to linearize plasmids)
                      • DI H2O
                        • PCR Tube
                          • Thermal Cycler
                            • Everything Important but DNA (It's a good master mix candidate)
                              • 5 μL T4 DNA Ligase
                              • 2 μL T4 DNA Ligase Buffer
                              • 1 μL Type IIS Restriction Enzyme (we have SapI and BsaI)
                              • 0.2 μL 100X or 1 μL 20X BSA (Bovine Serum Albumin)

                            Procedure

                            • Everything
                            1. Add the non-DNA components to a new PCR tube
                            1. Add an appropriate amount of DNA (75 ng per component is recommended)
                            1. Fill to 20 μL with DI H2O
                            1. For 1-4 fragments, incubate at 37°C for 1 hr and 55°C for 5 min. For 5-10 fragments, alternate between 37°C and 16°C for one minute each for 30 cycles, then incubate for 5 min at 55°C. For more than 10 fragments, consider going that extra mile and buying the optimized master mix.
                            1. Proceed directly to transfomation.
                            H2O2/Peroxidase (Amplex Red) Assay · Benchling

                            H2O2/Peroxidase (Amplex Red) Assay

                            Introduction

                            Used to detect presence of hydrogen peroxide (H2O2) or peroxidase activity in biological samples. Specifically used to detect the activity of lysyl oxidase in coupling monomers.

                            Materials

                              • H2O2/Peroxidase/Amplex Red Assay kit from Thermofisher
                              • 96 well plate
                              • plate reader

                            Procedure

                            • Prep for stock solutions
                            1. Let all reagents in the kit to completely warm to RT before opening.
                            1. 10 mM Amplex Red reagent stock soln: It is AIR SENSITIVE, use the reagent on the same day. Also light sensitive. Use on the same day it's prepared.
                            • Let Amplex Red reagent (Component A, blue cap) and DMSO (Component B, green cap) warm to room temp. Just before using, dissolve Amplex Red reagent into 60 uL of DMSO.
                            1. 1X Rxn buffer: Add 4 mL of 5x Reaction buffer (Component C, white cap) to 16 mL of dH2O.
                            1. 10 U/mL Horseradish peroxidase stock soln: Dissolve HRP (Component D, yellow cap) in 1.0 mL of 1x Rxn buffer.
                            • Divide any unused HRP stock soln into single use aliquots and store frozen at -20C.
                            1. 20 mM H2O2 working soln: Dilute ~3% H2O2 (Component E, red cap) into appropriate vol of 1X Rxn buffer to make 20 mM H2O2. Actual concentration of H2O2 is indicated on the label.
                            • 20 mM working solution eeds to be used within a few hours of prep
                            • H2O2 Assay
                            1. Prepare H2O2 standard curve. Dilute H2O2 containing samples in 1X Rxn buffer to produce H2O2 concentrations of 0-10 uM.
                            • Use volume of 50 uL for each rxn. Pipet into individual wells of microplate.
                            1. Prep working soln of 100 uM Amplex Red Reagent/HRP. Mix 50 uL of 10 mM Amplex Red reagent stock soln + 100 uL of 10 U/mL HRP stock soln, 4.85 mL of 1X Rxn buffer.
                            1. Begin rxn. Add50 uL of Amplex Red reagent/HRP working soln to each microplate well containing standards/controls/samples.
                            1. Incubate rxns at room temp for 30 mins, protected from light.
                            1. Use plate reader to detect fluorescence (excitation 530-560 nm, emission 590nm, absorbance 560nm).
                            1. For each point subtract value derived from no-H2O2 control
                            Inclusion Body Solubilization Reagent · Benchling

                            Inclusion Body Solubilization Reagent

                            Introduction

                            Recover insoluble proteins expressed in inclusion bodies (protein aggregates)

                            Materials

                              • B-PER Bacterial Protein Extraction Reagent
                              • Inclusion Body Solubilization Reagent

                            Procedure

                            • Protein solubilization
                            1. Estimate amount of inclusion body prep by subtracting weight of centifuge tube from total weight. Use 8 mL of Inclusion Body Solubilization Reagent per 1 g of wet inclusion body pellet.
                            1. Suspend pellet in appropriate amount of Inclusion Body Solubilization by vortexing or pipetting. Shake suspension of 30 min.
                            1. Remove cell debris by centrifugation at 27k x g (15k rpm) for 15 min.
                            1. Collect supernatant which contains the solubilized protein. For protein assay, use Coomassie Plus (Bradford) Protein Assay Kit. For SDS-PAGE analysis, remove denaturant by dialysis.
                            Large Scale Extraction · Benchling

                            Large Scale Extraction

                            Introduction

                            Get started by giving your protocol a name and editing this introduction.

                            Materials

                              • 1 g of wet cell paste or 250 mL bacterial culture with OD600 of ~2.0
                              • CelLytic B

                            Procedure

                            1. Collect bacterial cells that express protein of interest by centrifuging at 5k x g for 10 min.
                            1. Carefully remove the spent medium from the cell pellet. The cell pellet may be frozen or used fresh. Frozen cell pellet will give slightly higher yield of protein
                            1. Add CelLytic B at ratio of 10-20 mL per gram of cell paste. Mix well to completely resuspend the cells.
                            • Add lysozyme (final concentration = 0.2 mg/mL) to enhance cell lysis
                            • Benzonase (final amount of 50 units/mL) to decrease viscosity of solution.
                            • Protease inhibitors to prevent proteolytic degradation
                            1. Incubate extraction suspension with vortexing/shaking at room temperature for 10-15 minutes to fully extract soluble proteins from cells
                            1. After cell extraction, centrifuge the extract at 16k x g for 10 mins to pellet insoluble material.
                            1. Carefully remove supernatant containing soluble protein fraction. Another round of extraction will yield more soluble protein if required - but will result in more dilute soluble protein sample
                            1. Analyze supernatant and insoluble fraction by SDS-PAGE and/or Western blot to determine which fraction contains protein of interest. For SDS-PAGE use 5-15 uL of each sample for gel.
                            Ligating Complementary Strands (Gibson) · Benchling

                            Ligating Complementary Strands (Gibson)

                            Introduction

                            Ligating complementary strands before Gibson Assembly and transformation.

                            Materials

                              • 5ul T4 DNA ligase buffer (in freezer in 348 with other restriction enzymes)
                              • 43ul DI water
                              • 1ul forward strands (undiluted)
                              • 1ul reverse strands (undiluted)
                              • 2.5ul 1M NaCl
                              • PCR tubes

                            Procedure

                            • Ligation
                            1. Thaw ligase buffer in hand and vortex, then put immediately on ice.
                            1. Mix materials in PCR tubes with a total volume of 52.5ul
                            1. Set at 95˚C and cool at 0.1˚C on Jessica's thermocycler for 19 minutes
                            1. Dilute 1:10 if doing Gibson
                            1. Do Gibson protocol
                            Liquid Culture · Benchling

                            Liquid Culture

                            Introduction

                            Liquid culture of cells

                            Materials

                              • Antibiotic
                              • LB broth
                              • Cell culture

                            Procedure

                            • Liquid Culture
                            1. Add 5ml LB broth to 15ml tube.
                            1. Add 5ul antibiotic.
                            1. Add +/- 1ul/scrape of cells.
                            1. Shake up and incubate at 37˚C at 180rpm.
                            Lumio Green Detection · Benchling

                            Lumio Green Detection

                            Introduction

                            It's pretty great. You can do this even before inducing.

                            Materials

                            Procedure

                            Making LB Agar Plates · Benchling

                            Making LB Agar Plates

                            Introduction

                            Making LB agar plates.

                            Materials

                              • Agar powder
                              • LB broth
                              • 1ml antibiotic/L of liquid

                            Procedure

                            • Making Broth
                            1. Add agar powder (15g/L) to LB broth.
                            1. Autoclave.
                            1. Cool to 55˚C then add antibiotic. Pour into petri dishes.
                            1. Allow to harden and store inverted at 4˚C in the dark (antibiotic is light sensitive).
                            1. If agar solidifies too early, possible to microwave it but it may explode.
                            Nanodrop Reader · Benchling

                            Nanodrop Reader

                            Introduction

                            Reading DNA concentration with a Nanodrop.

                            Materials

                              • Tube of DNA

                            Procedure

                            • Reading DNA
                            1. Read blank of 1ul elution buffer.
                            1. Run each sample, name at Sample ID, hit measure.
                            1. Want 260/280 + 260/280 ~ 2.
                            1. Record concentrations on stored tubes.
                            Native PAGE · Benchling

                            Native PAGE

                            Introduction

                            Actually this is on a card I don't possess, should be in the lab though. I'll look it up at some point soon. Good luck Anna!

                            Materials

                            Procedure

                            PCR · Benchling

                            PCR

                            Introduction

                            Get started by giving your protocol a name and editing this introduction. *Primer Tm 50˚C target*

                            Materials

                              • Taq master mix
                              • Forward primer
                              • Reverse primer
                              • DI water
                              • Template DNA
                              • PCR tubes

                            Procedure

                            • Polymerase Chain Reaction
                            1. In a PCR tube, mix:
                            • 12.5ul Taq master mix (green)
                            • 1ul 10uM forward primer (1:20 dilution may be needed)
                            • 1ul 10uM reverse primer (1:20 dilution may be needed)
                            • 10.5ul super DI water
                            • Touch of template DNA
                            1. Maintain all on ice throughout process.
                            1. Gently mix tube.
                            1. Place in thermocycler and cycle at:
                            • 94˚C (30s)
                            • Circle 30 times: 94˚C (15-30s), 45-68˚C (15-60s), 68˚C (1min/kb)
                            • 68˚C (5min)
                            1. Final hold 4-10˚C.
                            Phosphorylation and Ligation of Repetitive DNA Building Blocks · Benchling

                            Phosphorylation and Ligation of Repetitive DNA Building Blocks

                            Introduction

                            Use this protocol to phosphorylate and repetitive DNA buidling blocks, and ligate them into randomized long repetitive strands.

                            Materials

                            • Materials per Tube (Phosphorylation) Volume
                              • DNA building block mixture (80 µM) 5 µl
                              • Ligase 10x Buffer 2 µl
                              • DI Water 11.5 µl
                              • T4 Polynucleotide Kinase (PNK) 1.5 µl
                            • Total Volume (Phosphorylation) 20 µl
                              • Materials per Tube (Ligation)
                                • Phosphorylation mix (above) 20 µl
                                • T4 DNA Ligase 1 µl
                              • Total Volume (Phosphorylation) 21 µl

                                Procedure

                                • Phosphorylation
                                1. Place all materials on ice to thaw. If needed, thaw Ligase 10x Buffer in hands and vortex until all white precipitate has disolved. Place back on ice for 2 min before adding to rxn mixture.
                                1. Add materials listed above with desired 80 µM DNA building block mix to PCR tube
                                1. Incubate in thermal cycler at 37°C for 1 hr
                                • Ligation
                                1. Add 1 µl T4 DNA Ligase to each tube -- vortex and spin down to mix if
                                1. Incubate at 16°C overnight
                                Scanning a Gel · Benchling

                                Scanning a Gel

                                Introduction

                                Scanning gels.

                                Materials

                                  • Gel to read

                                Procedure

                                1. Carefully remove gel from box and place on scanner.
                                1. Select area on scanner.
                                1. Run scanner.
                                Sequencing Order · Benchling

                                Sequencing Order

                                Introduction

                                https://www.elimbio.com/

                                Materials

                                  • 1 uL primer_F (VF) diluted 1:20
                                  • 1 1uL primer_R (VR) diluted 1:20
                                  • 30ng DNA
                                  • 15 uL total volume water

                                Procedure

                                Small/Trial Scale Extraction · Benchling

                                Small/Trial Scale Extraction

                                Introduction

                                Determine the fraction in which the protein of interest will be found

                                Materials

                                  • Bacterial culture OD600 0.5-1.0
                                  • CelLytic B

                                Procedure

                                1. Use 1.5 mL of bacterial culture with OD600 of 0.5-1.0 and centrifuge the cells at full speed/7k for 2 min.
                                1. Remove spun medium and resuspend the cell pellet in 0.4 CelLytic B.
                                1. Carefully remove the soluble protein fraction from the cell debris. Additional extractions may be performed if required, but will make your soluble protein sample more dilute.
                                1. Analyze supernatant and insoluble fraction by SDS-PAGE and/or Western blot to determine which fraction contains the protein of interest.Fir SDS-PAGE, use 5-15 uL of each sample for gel.
                                T7 Transformations · Benchling

                                T7 Transformations

                                Introduction

                                High efficiency T7 protocol.

                                Materials

                                  • See general transformation materials

                                Procedure

                                • Transformations
                                1. Thaw cells on ice for 10min.
                                1. Add 1-5ul contianing 1pg-100ng plasmid DNA (eg from Gibson assembly).
                                1. Place mixture on ice for 30min. Do not mix.
                                1. Heat shock at 42˚C for 10s exactly.
                                1. Place on ice for 5min.
                                1. Pipette 950ul room temp SOC.
                                1. Place at 37˚C for 60min, shaking at 250rpm.
                                1. Warm selection plates to 37˚C.
                                1. Mix cells and perform dilutions.
                                1. Spread 50-100ul of cell dilution onto plates. Incubate overnight at 37˚C.
                                Transformation: Chemically Competent Cells · Benchling

                                Transformation: Chemically Competent Cells

                                Introduction

                                Transformation protocol for chemically competent cells.

                                Materials

                                  • SOC (super optimal broth with catabolite repression)
                                  • NED 5-alpha competent E.coli cells (Half a stock)
                                  • Purified DNA
                                  • Agar plates with chloramphenicol

                                Procedure

                                • Transformation
                                1. Thaw NEB 5-alpha competent E.coli on ice for 10 minutes (Half a stock is fine, half each subsequent amount)
                                1. Add 1-5ul of 1pg-100ng plasmid DNA to cell mixture. Flick tube to mix, do not vortex.
                                1. Place on ice for 30min. Do not mix.
                                1. Heat shock at 42˚C for 30s.
                                1. Place on ice for 5min.
                                1. Pipette 950ul room temp SOC into mixture (475 if using half stock).
                                1. Place at 37˚C for 60 minutes, shaking at 250rpm.
                                1. Perform several 10-fold dilutions (1:10, 1:100) of cells.
                                1. Plate 50-100ul of each dilution onto an antibiotic selection plate.
                                1. Incubate overnight at 37˚C.
                                Transformation: Electrocompetent Cells · Benchling

                                Transformation: Electrocompetent Cells

                                Introduction

                                Transformation protocol for electrocompetent cells. To make electrocompetent cells, refer to: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939052/.

                                Materials

                                  • Agar plates with chloramphenicol
                                  • NED 5-alpha competent E.coli cells
                                  • SOC media
                                  • Purified DNA

                                Procedure

                                • Transformation
                                1. Place electroporation cuvettes, microcentrifuge, and cuvette holder on ice.
                                1. Thaw NEB 5-alpha electrocompetent E.coli on ice for 10min.
                                1. Transfer 25ul of cells and 1ul DNA into microcentrifuge tube.
                                1. Transfer mixture to cuvette. Make sure all parts are cold to avoid cells exploding.
                                1. Electroporate with the following conditions:
                                1. Add 975ul SOC in cell box to cuvette, mixing gently, then place in round bottom culture tube.
                                1. Incubate at 37˚C, shaking at 250rpm for 1hr.
                                1. Dilute cells (1:10, 1:100) and spread 100-200ul onto antibiotic selection plate.
                                1. Incubate cells overnight at 37˚C.