View source for Team:Toronto/Experiment-iGEM-Competent-Cell-Test-Kit

<html>
<!-- ####################################################### -->
<!-- #  This html was produced by the igemwiki generator   # -->
<!-- #  https://github.com/igemuoftATG/generator-igemwiki  # -->
<!-- ####################################################### -->

<!-- repo for this wiki: https://github.com/igemuoftATG/wiki2016 -->
</html>
{{Toronto/head}}
<html>

<div id="navigation">
<ul>
<li><a href="https://2016.igem.org/Team:Toronto"><span>home</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Achievements"><span>achievements</span></a></li>
</li>
<li><a href="#"><span>team</span></a>
<ul>
<li><a href="https://2016.igem.org/Team:Toronto/Team"><span>team</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Collaborations"><span>collaborations</span></a></li>
</li>
</ul>
<li class="active"><a href="#"><span>project</span></a>
<ul>
<li><a href="https://2016.igem.org/Team:Toronto/Description"><span>description</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Design"><span>design</span></a></li>
</li>
<li class="active"><a href="https://2016.igem.org/Team:Toronto/Experiments"><span>experiments</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Proof"><span>proof</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Demonstrate"><span>demonstrate</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Results"><span>results</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Notebook"><span>notebook</span></a></li>
</li>
</ul>
<li><a href="#"><span>parts</span></a>
<ul>
<li><a href="https://2016.igem.org/Team:Toronto/Parts"><span>parts</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Basic_Part"><span>basic_part</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Composite_Part"><span>composite_part</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Part_Collection"><span>part_collection</span></a></li>
</li>
</ul>
<li><a href="https://2016.igem.org/Team:Toronto/Safety"><span>safety</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Attributions"><span>attributions</span></a></li>
</li>
<li><a href="#"><span>human_practices</span></a>
<ul>
<li><a href="https://2016.igem.org/Team:Toronto/Human_Practices"><span>human_practices</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/HP-Silver"><span>silver</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/HP-Gold"><span>gold</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/HP-Impact"><span>impact</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Integrated_Practices"><span>integrated_practices</span></a></li>
</li>
</ul>
<li><a href="#"><span>awards</span></a>
<ul>
<li><a href="https://2016.igem.org/Team:Toronto/Software"><span>software</span></a></li>
</li>
<li><a href="https://2016.igem.org/Team:Toronto/Model"><span>model</span></a></li>
</li>
</ul>
</ul>
</div>
<div class="content">



<html lang="en">
<head>
<meta charset="utf-8"/>
<title>iGEM Competent Cell Test Kit · 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="pv1242-title" data-index="0">iGEM Competent Cell Test Kit</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="pv1242-intro" data-index="0">The following is coped directly from: http://parts.igem.org/Help:Competent_Cell_Test_Kit

This protocol is to be performed directly after the generation of competent cells.

The kit includes five vials of purified DNA from <a href="http://parts.igem.org/Part:BBa_J04450" rel="nofollow" target="_blank">BBa_J04450 </a>(RFP construct) in plasmid backbone pSB1C3. Each vial contains DNA at a different concentration: 50pg/ul, 20pg/ul, 10pg/ul, 5pg/ul, 0.5pg/ul. Perform transformations with each of these to determine how efficient your competent cells are.

<b><i>Note</i></b><i>:</i> Transformations performed with ligation products usually do not yield as many colonies because of the low DNA concentration in the ligation mixture. This means that you may see different results doing this test than you will at the end of the 3A Assembly protocol, or any other ligation.
</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="m1243" data-index="0">70% ethanol</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1244" data-index="1">Paper towels</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1245" data-index="2">Lab marker / Sharpie</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1246" data-index="3">Container for ice</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1247" data-index="4">Ice</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1248" data-index="5">Competent cell aliquot(s)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1249" data-index="6"><b>Competent Cell Test Kit</b> (previously called, <i>Transformation Efficiency Kit</i>)</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1250" data-index="7">Agar plates with chloramphenicol</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1251" data-index="8">Waterbath (or hot water source and thermometer</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1252" data-index="9">Incubator</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1253" data-index="10">SOC media</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1254" data-index="11">Sterile glass beads or inoculating loop</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1255" data-index="12">Pipettor</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m1256" data-index="13">Pipette tips</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="s1257" data-index="0"><i><span style="color:#344043"><span style="background-color:#FFFFFF">Estimated time: 30 minutes active, 2.5 hours incubation</span></span></i></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1257" 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="s1258" data-index="1">Spin down the DNA tubes from the Competent Cell Test Kit/Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient. <i>Note:</i> There should be 50 µL of DNA in each tube sent in the Kit.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1258" 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="s1259" data-index="2">Thaw competent cells on ice. Label one 2.0ml microcentrifuge tube for each concentration and then pre-chill by placing the 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-s1259" 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="s1260" data-index="3">Pipet 1 µL of DNA into each microcentrifuge tube. For each concentration, use a separate tube.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1260" 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="s1261" data-index="4">Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1261" 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="s1262" data-index="5">Heat-shock the cells by placing into the waterbath for 1 minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1262" 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="s1263" data-index="6">Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1263" 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="s1264" data-index="7">Add 200 µL of SOC media per tube, and incubate at 37°C for 2 hours. Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1264" 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="s1265" data-index="8">Pipet 20 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Do triplicates (3 each) of each tube if possible, so you can calculate an average colony yield. Incubate at 37°C overnight or approximately 16 hours. Position the plates so the agar side is facing up, and the lid is facing down.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1265" 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="s1266" data-index="9">Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1266" 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="s1267" data-index="10">␥ (colonies on plate) / ng of DNA plated x 1000ng/µg</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="s1268" data-index="11"><span style="color:#344043"><span style="background-color:#FFFFFF">␥ </span></span><i>Note: The measurement "ng of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:</i></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="s1269" data-index="12"><span style="color:#344043"><span style="background-color:#FFFFFF">␥ </span></span>1 µL x concentration of DNA (refer to vial) x (volume plated / total reaction volume)</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="s1270" data-index="13">Results</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1270" data-index="13"></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="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1271" data-index="14">Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 
cfu/µg DNA, where "cfu" means "colony-forming unit" and is a measurement
 of cells.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1271" 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="11"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1272" data-index="15">Here are some sample results.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s1272" 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-table"><div class="mediocre-tableEditable" data-formattable="true" data-index="16" data-subindex="cell" style="margin-left:0;margin-right:0;"><div></div><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="hiddenFocusEditable editable" contenteditable="true" data-cid="s1273-table" data-formattable="true" data-index="16" data-subindex="table"></div><div class="mediocre-tableEditable-scrollArea"><div class="mediocre-tableEditable-tableWrapper" style="padding-left:0;"><div class="mediocre-tableEditable-border multi-editable" style="left:-10px;width:665px;"></div><table class="mediocre-tableEditable-table u-small-spacing-right" style="top:0;width:645px;"><colgroup><col style="width:45px;"></col><col style="width:100px;"></col><col style="width:100px;"></col><col style="width:100px;"></col><col style="width:100px;"></col><col style="width:100px;"></col><col style="width:100px;"></col></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-maximizeButton"><span class="glyphicon glyphicon-arrow-up mediocre-tableEditable-maximizeIcon" icon="arrow-up"></span></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-numWrapper"><span class="mediocre-tableEditable-axisCell-num">A</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-numWrapper"><span class="mediocre-tableEditable-axisCell-num">B</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-numWrapper"><span class="mediocre-tableEditable-axisCell-num">C</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-numWrapper"><span class="mediocre-tableEditable-axisCell-num">D</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-numWrapper"><span class="mediocre-tableEditable-axisCell-num">E</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div class="mediocre-tableEditable-axisCell-numWrapper"><span class="mediocre-tableEditable-axisCell-num">F</span></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-numWrapper"><span class="mediocre-tableEditable-axisCell-num">1</span></div></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-0-0" data-namespace="table-ns3280" style="color:#344043;"><span>DNA concentration</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-0-1" data-namespace="table-ns3280" style="color:#344043;"><span>0.5pg/ul</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-0-2" data-namespace="table-ns3280" style="color:#344043;"><span>5pg/ul</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-0-3" data-namespace="table-ns3280" style="color:#344043;"><span>10pg/ul</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-0-4" data-namespace="table-ns3280" style="color:#344043;"><span>20pg/ul</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-0-5" data-namespace="table-ns3280" style="color:#344043;"><span>50pg/ul</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div class="mediocre-tableEditable-axisCell-numWrapper"><span class="mediocre-tableEditable-axisCell-num">2</span></div></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-1-0" data-namespace="table-ns3280" style="color:#344043;"><span># of colonies</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-1-1" data-namespace="table-ns3280" style="color:#344043;"><span>10 - 20</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-1-2" data-namespace="table-ns3280" style="color:#344043;"><span>120 - 170</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-1-3" data-namespace="table-ns3280" style="color:#344043;"><span>280 - 360</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-1-4" data-namespace="table-ns3280" style="color:#344043;"><span>480 - 802</span></td><td class="cell is-text is-alignCenter" data-cid="table-s1273-1-5" data-namespace="table-ns3280" style="color:#344043;"><span>500 - 1000+</span></td></tr></tbody></table></div></div><div class="mediocre-tableEditable-titleBarWrapper" style="left:0;"><div class="mediocre-tableEditable-titleBar"><div class="u-ellipsis-text mediocre-tableEditable-tableName u-pointer">Table1</div><div class="table-floater-wrapper"></div></div></div></div></div><div class="mediocre-item is-image"><div class="image-editable"><form class="add-image-form"><input accept="image/*" class="image-input hidden" type="file"/><div class="upload-wrapper multi-editable"><img class="img" src="https://main-benchling.s3-external-1.amazonaws.com/proto-image/wlOveBSUmj8W0gaosNbr4k3u1vOh6Aekwkn9CKaR/competent cell test kit.png"/></div><div class="progress-bar" style="width:0%;"></div></form></div></div></div></div></div></div></div></div></div></div></div></div>
</body>
</html>




</div>
</html>
{{Toronto/footer}}