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{{WashU_StLouis}} | {{WashU_StLouis}} | ||
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<div class="column full_size"> | <div class="column full_size"> | ||
− | < | + | <div class = "title2"></div> |
− | < | + | <!--<div class="action"><img src="https://static.igem.org/mediawiki/2016/d/d9/T--WashU_StLouis--Action.png" style="width:100%;"></div>--> |
− | </ | + | <div class = "speech"><img src="https://static.igem.org/mediawiki/2016/0/05/T--WashU_StLouis--Speech.png" style="width:100%;"></div> |
+ | <h1 class="actionwords">Experiments</h1> | ||
+ | <h2 class="speechwords"> Check out the protocols we used throughout our project</h2> | ||
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<h3> Cloning </h3> | <h3> Cloning </h3> | ||
− | <p> | + | <p style= " text-align: center;"> We used these protocols to create plasmids with our genes of interest from genomic DNA, and transform those plasmids into <i>E. coli</i> </p> |
</br> | </br> | ||
<button class="protocol-accordion">Genomic DNA Extraction</button> | <button class="protocol-accordion">Genomic DNA Extraction</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | < | + | <ol> |
− | + | <li>Centrifuge incoulated cell culture for 15 minutes at 5000g</li> | |
− | <li>Centrifuge | + | |
<li>Discard supernatant and resuspend in 1 mL of cloning water</li> | <li>Discard supernatant and resuspend in 1 mL of cloning water</li> | ||
<li>Transfer solution into bashing bead lysis/filtration tube</li> | <li>Transfer solution into bashing bead lysis/filtration tube</li> | ||
Line 107: | Line 37: | ||
<li>Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g</li> | <li>Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g</li> | ||
<li>Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once</li> | <li>Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once</li> | ||
− | <li>Add 200 | + | <li>Add 200 μL of fungal/bacterial DNA wash buffer to column, spin for 1 minute at 10,000g, and repeat 3 more times</li> |
− | <li>Transfer column to a 1.5 mL microcentrifuge tube and add 30 | + | <li>Transfer column to a 1.5 mL microcentrifuge tube and add 30 μL of cloning water</li> |
− | <li>Let sit for 4 minutes, then spin for 4 minutes at 10, | + | <li>Let sit for 4 minutes, then spin for 4 minutes at 10,000g</li> |
<li>Measure concentration of gDNA and label tube</li> | <li>Measure concentration of gDNA and label tube</li> | ||
− | </ | + | </ol> |
</div> | </div> | ||
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<button class="protocol-accordion">PCR</button> | <button class="protocol-accordion">PCR</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
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− | < | + | <ol> |
<li>Add the following to a single PCR tube:</li> | <li>Add the following to a single PCR tube:</li> | ||
<ul> | <ul> | ||
− | <li>20-21.5 | + | <li>20-21.5 μL Cloning Water (depending on type of amplicon)</li> |
− | <li>10 | + | <li>10 μL of Betaine</li> |
− | <li>10 | + | <li>10 μL of 5x High-Fidelity Buffer </li> |
− | <li>4 | + | <li>4 μL DMSO</li> |
− | <li>2.5 | + | <li>2.5 μL 10 uM combined forward/reverse primers</li> |
− | <li>1 | + | <li>1 μL DNTPs</li> |
− | <li>0.5 | + | <li>0.5 μL of plasmid or 2 μL of gDNA</li> |
− | <li>0.5 | + | <li>0.5 μL of Phusion Polymerase (kept on ice)</li> |
</ul> | </ul> | ||
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<ul> | <ul> | ||
− | <li>Initial Denaturation | + | <li>Initial Denaturation 98°C 30 sec</li> |
<li>25-35 cycles</li> | <li>25-35 cycles</li> | ||
<ul> | <ul> | ||
− | <li>98 C 5-10 sec</li> | + | <li>98°C 5-10 sec</li> |
− | <li>45-72 C (at annealing temp) 10-30 sec</li> | + | <li>45-72°C (at annealing temp) 10-30 sec</li> |
− | <li>72 C 15-30 sec/(length of amplicon in kb)</li> | + | <li>72°C 15-30 sec/(length of amplicon in kb)</li> |
</ul> | </ul> | ||
− | <li>Final Extension 72 C 5-10 minutes</li> | + | <li>Final Extension 72°C 5-10 minutes</li> |
− | <li>Hold at | + | <li>Hold at 4°C</li> |
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− | + | ||
</ul> | </ul> | ||
+ | <li>Store at 4°C until use</li> | ||
+ | </ol> | ||
</div> | </div> | ||
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<div class="protocol-panel"> | <div class="protocol-panel"> | ||
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− | < | + | <ol> |
<li>Measure 1 gram of Agarose per 100 mL TAE buffer (Tris base, acetic acid and EDTA) </li> | <li>Measure 1 gram of Agarose per 100 mL TAE buffer (Tris base, acetic acid and EDTA) </li> | ||
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<ul> | <ul> | ||
− | <li>We generally made gels with 75 - | + | <li>We generally made gels with 75 - 125 mL of TAE</li> |
</ul> | </ul> | ||
<li>Microwave in a covered erlenmeyer flask for 120 seconds</li> | <li>Microwave in a covered erlenmeyer flask for 120 seconds</li> | ||
− | <li>Add 1 | + | <li>Add 1 μL of SYBR Safe or Ethidium Bromide per 20 mL of TAE</li> |
<li>Pour into gel casing</li> | <li>Pour into gel casing</li> | ||
− | <li>Remove bubbles with a | + | <li>Remove bubbles with a pipette tip</li> |
<li>Insert well comb</li> | <li>Insert well comb</li> | ||
<li>Allow to set (~20 minutes)</li> | <li>Allow to set (~20 minutes)</li> | ||
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<li>Remove well comb</li> | <li>Remove well comb</li> | ||
<li>Place gel in electrophoresis apparatus with the wells by the cathode (black)</li> | <li>Place gel in electrophoresis apparatus with the wells by the cathode (black)</li> | ||
<li>Fill apparatus with TAE buffer so that the gel is completely submerged</li> | <li>Fill apparatus with TAE buffer so that the gel is completely submerged</li> | ||
− | <li>Load wells with 10-60 | + | <li>Load wells with 10-60 μL of 5 (PCR product): 1 (6x Loading Dye)</li> |
− | <li>Load | + | <li>Load 10 μL of ladder into first and last wells</li> |
− | <li>Run at 125 | + | <li>Run at 125-130 V for 25-30 minutes</li> |
− | </ | + | </ol> |
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</div> | </div> | ||
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<button class="protocol-accordion">Gel Extraction</button> | <button class="protocol-accordion">Gel Extraction</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | <p> | + | <p style = "padding: 0px 0px 0px 50px;">(Adapted from Zymo Research Zymoclean™ Gel DNA Recovery Kit)</p> |
− | < | + | <ol> |
<li>Cut band from gel while minimizing time under UV light</li> | <li>Cut band from gel while minimizing time under UV light</li> | ||
<li>Place gel in a microcentrifuge tube with 3x volume of Agarose Dissolving Buffer</li> | <li>Place gel in a microcentrifuge tube with 3x volume of Agarose Dissolving Buffer</li> | ||
− | <li>Heat at 55 C until gel dissolves (~20 minutes)</li> | + | <li>Heat at 55°C until gel dissolves (~20 minutes)</li> |
<li>Add to gel purification filter placed inside a collection tube</li> | <li>Add to gel purification filter placed inside a collection tube</li> | ||
− | <li>Centrifuge for 1 minute at 16,000 | + | <li>Centrifuge for 1 minute at 16,000 g and discard flow-through</li> |
− | <li>Add 200 | + | <li>Add 200 μL of DNA wash buffer, centrifuge for 1 minute at 16,000 g, discard flow-through, and repeat once</li> |
− | <li>Centrifuge for 2 minutes at 16,000 | + | <li>Centrifuge for 2 minutes at 16,000 g and discard flow-through</li> |
− | <li>Place filter in 1.5 mL microcentrifuge and add 20-30 | + | <li>Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water</li> |
− | <li>Wait for 4 minutes, then spin for 4 minutes at 16,000 | + | <li>Wait for 4 minutes, then spin for 4 minutes at 16,000 g</li> |
<li>Measure concentrations and label</li> | <li>Measure concentrations and label</li> | ||
− | </ | + | </ol> |
</div> | </div> | ||
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<button class="protocol-accordion">DNA Purification</button> | <button class="protocol-accordion">DNA Purification</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | <p> | + | <p style = "padding: 0px 0px 0px 50px;">(Adapted from Zymo Research DNA Clean & Concentrator™-5 Kit)</p> |
− | + | ||
− | < | + | <ol> |
− | <li>Add 50 | + | <li>Add 50 μL DNA and 250 μL DNA Binding Buffer to a spin column in a collection tube</li> |
<li>Centrifuge for 30 seconds at Xg and discard flow-through</li> | <li>Centrifuge for 30 seconds at Xg and discard flow-through</li> | ||
− | <li>Add 200 | + | <li>Add 200 μL of DNA wash buffer, centrifuge for 30 seconds at 16,000g, discard flow-through, and repeat once |
− | <li>Place filter in 1.5 mL microcentrifuge and add 20-30 | + | <li>Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water</li> |
− | <li>Wait for 4 minutes, then spin for 4 minutes at 16, | + | <li>Wait for 4 minutes, then spin for 4 minutes at 16,000g</li> |
<li>Measure concentrations and label</li> | <li>Measure concentrations and label</li> | ||
− | </ | + | </ol> |
</div> | </div> | ||
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<button class="protocol-accordion">Golden Gate Assembly</button> | <button class="protocol-accordion">Golden Gate Assembly</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <ol> | |
− | + | <li>In a PCR tube, combine:</li> | |
− | + | <ul> | |
− | + | <li>0.4 μL Cutsmart Buffer</li> | |
− | + | <li>0.4 μL DpnI</li> | |
+ | <li>plasmid backbone (either post PCR or gel extract)</li> | ||
+ | </ul> | ||
+ | <li>Incubate at 37°C for 1 hour</li> | ||
+ | <li>DNA purification for post-DpnI mix to retrieve plasmid backbone</li> | ||
+ | <li>In a PCR tube, combine:</li> | ||
+ | <ul> | ||
+ | <li>100 ng of plasmid backbone</li> | ||
+ | <li>Equimolar amount of each assembly piece</li> | ||
+ | <li>1.5 μL of restriction enzyme buffer</li> | ||
+ | <li>1 μL of ligase buffer</li> | ||
+ | <li>1 μL of Type IIs restriction enzyme</li> | ||
+ | <li>1 μL of T4 ligase</li> | ||
+ | <li>Cloning water to equal a total of 15 μL</li> | ||
+ | </ul> | ||
+ | <li>Run in a thermocycler for the following times/temperatures:</li> | ||
+ | <ul> | ||
+ | <li>50 cycles</li> | ||
+ | <ul> | ||
+ | <li>3 minutes at 37°C</li> | ||
+ | <li>4 minutes at 16°C</li> | ||
+ | </ul> | ||
+ | <li>1 cycle</li> | ||
+ | <ul> | ||
+ | <li>5 minutes at 50°C</li> | ||
+ | <li>20 minutes at 80°C</li> | ||
+ | </ul> | ||
+ | <li>Hold at 4°C</li> | ||
+ | </ul> | ||
+ | <li>Transform into competent cells and/or store at -20°C</li> | ||
+ | </ol> | ||
+ | |||
</div> | </div> | ||
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<button class="protocol-accordion">Transformation</button> | <button class="protocol-accordion">Transformation</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <p>Electroporation</p> | |
− | + | <ol> | |
− | < | + | |
<li>Defrost competent cells on ice for 5 minutes</li> | <li>Defrost competent cells on ice for 5 minutes</li> | ||
− | <li>Pipet 1.5 | + | <li>Pipet 1.5 μL plasmid to the tube of competent cells</li> |
<li>Pipet the mixture into an electroporation cuvette</li> | <li>Pipet the mixture into an electroporation cuvette</li> | ||
<ul> | <ul> | ||
<li>Avoid touching the metal plate of the cuvette and introducing bubbles</li> | <li>Avoid touching the metal plate of the cuvette and introducing bubbles</li> | ||
</ul> | </ul> | ||
− | <li>Shock | + | <li>Shock</li> |
− | <li>Quickly add 500 | + | <li>Quickly add 500 μL of Lysogeny Broth (LB)</li> |
− | <li>Incubate in a culture tube for 1 hour at 37 C</li> | + | <li>Incubate in a culture tube for 1 hour at 37°C</li> |
<li>Pipet desired amount of solution onto a plate and spread evenly</li> | <li>Pipet desired amount of solution onto a plate and spread evenly</li> | ||
<li>Allow solution to dry and incubate for 16-18 hours</li> | <li>Allow solution to dry and incubate for 16-18 hours</li> | ||
− | <li>Wrap plate in parafilm and store at 4 C</li> | + | <li>Wrap plate in parafilm and store at 4°C</li> |
− | </ | + | </ol> |
− | Chemical | + | <p>Chemical Transdormation</p> |
− | < | + | <ol> |
− | <li>Thaw 100 | + | <li>Thaw 100 μL of competent cells on ice</li> |
<li>Add 100 ng of plasmid to cells</li> | <li>Add 100 ng of plasmid to cells</li> | ||
<li>Incubate on ice for 20-30 minutes</li> | <li>Incubate on ice for 20-30 minutes</li> | ||
− | <li>Heat shock the cells for 60 seconds at 42 C</li> | + | <li>Heat shock the cells for 60 seconds at 42°C</li> |
<li>Return the cells to ice for 2 minutes</li> | <li>Return the cells to ice for 2 minutes</li> | ||
− | <li>Add 300 | + | <li>Add 300 μL of LB or SOB medium</li> |
− | <li>Incubate in a culture tube for 1 hour at 37 C</li> | + | <li>Incubate in a culture tube for 1 hour at 37°C</li> |
<li>Pipet desired amount of solution onto a plate and spread evenly</li> | <li>Pipet desired amount of solution onto a plate and spread evenly</li> | ||
<li>Allow solution to dry and incubate for 16-18 hours</li> | <li>Allow solution to dry and incubate for 16-18 hours</li> | ||
− | <li>Wrap plate in parafilm and store at 4 C</li> | + | <li>Wrap plate in parafilm and store at 4°C</li> |
− | </ | + | </ol> |
</div> | </div> | ||
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<button class="protocol-accordion">Plasmid Miniprep</button> | <button class="protocol-accordion">Plasmid Miniprep</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | <p> | + | <p style = "padding: 0px 0px 0px 50px;">Adapted from Zymo Research’s Zyppy Plasmid Miniprep kit</p> |
− | Adapted from Zymo Research’s Zyppy Plasmid Miniprep kit | + | <ol> |
− | + | <li>Spin down cells for 15 minutes at 3200g</li> | |
− | < | + | <li>Discard flow-through and resuspend in 600 μL of cloning water</li> |
− | <li>Spin down cells for 15 minutes at | + | |
− | <li>Discard flow-through and resuspend in 600 | + | |
<li>Transfer to a 1.5 mL microcentrifuge tube</li> | <li>Transfer to a 1.5 mL microcentrifuge tube</li> | ||
− | <li>Add 100 | + | <li>Add 100 μL of 6x Lysis Buffer and invert</li> |
− | <li>Add 300 | + | <li>Add 300 μL of cold Neutralization Buffer and mix thoroughly</li> |
− | <li>Microcentrifuge for 4 minutes at 16, | + | <li>Microcentrifuge for 4 minutes at 16,000g</li> |
<li>Pour supernatant into a miniprep filter placed in a collection tube</li> | <li>Pour supernatant into a miniprep filter placed in a collection tube</li> | ||
− | <li>Microcentrifuge for 1 minute at 16, | + | <li>Microcentrifuge for 1 minute at 16,000g and discard flow-through</li> |
− | <li>Add 200 | + | <li>Add 200 μL of Endo-wash buffer</li> |
<li>Microcentrifuge for 30 seconds at 16,000g and discard flow-through</li> | <li>Microcentrifuge for 30 seconds at 16,000g and discard flow-through</li> | ||
− | <li>Add 400 | + | <li>Add 400 μL of Zyppy Wash buffer</li> |
<li>Microcentrifuge for 30 seconds at 16,000g and discard flow-through</li> | <li>Microcentrifuge for 30 seconds at 16,000g and discard flow-through</li> | ||
<li>Microcentrifuge for 2 minutes at 16,000g and discard flow-through</li> | <li>Microcentrifuge for 2 minutes at 16,000g and discard flow-through</li> | ||
− | <li>Place filter in 1.5 mL microcentrifuge and add 20-30 | + | <li>Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water</li> |
− | <li>Wait for 4 minutes, then spin for 4 minutes at 16, | + | <li>Wait for 4 minutes, then spin for 4 minutes at 16,000g</li> |
<li>Measure concentrations and label</li> | <li>Measure concentrations and label</li> | ||
− | </ | + | </ol> |
</div> | </div> | ||
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<button class="protocol-accordion">Inoculation</button> | <button class="protocol-accordion">Inoculation</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <ol> | |
− | + | <li>Add 4 mL of Lysogeny broth (LB) and 4 μL of each needed antibiotic to a culture tube</li> | |
− | <li>Add 4 mL of Lysogeny broth (LB) and 4 | + | |
<li>Add cells to the culture tube</li> | <li>Add cells to the culture tube</li> | ||
<ul> | <ul> | ||
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<li>If using a plate, carefully scrape a single colony with a pipette tip</li> | <li>If using a plate, carefully scrape a single colony with a pipette tip</li> | ||
</ul> | </ul> | ||
− | <li>Grow at 37 C for 16-18 hours</li> | + | <li>Grow at 37°C for 16-18 hours</li> |
− | </ | + | </ol> |
</div> | </div> | ||
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<h3> BioBrick </h3> | <h3> BioBrick </h3> | ||
− | <p> | + | <p style= " text-align: center;"> We used these protocols to manipulate BioBricks provided in the iGEM distribution kit and to create our own BioBricks from experimentally tested genes</p> |
</br> | </br> | ||
<button class="protocol-accordion">Digestion</button> | <button class="protocol-accordion">Digestion</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <ol> | |
− | + | <li>Add the following to a PCR tube</li> | |
− | + | <ul> | |
− | + | <li>29 μL of miniprepped plasmid</li> | |
− | + | <li>11 μL Cloning water</li> | |
+ | <li>5 μL of NEB Buffer (depends on REs used)</li> | ||
+ | <li>5 μL total of Restriction Enzymes (SpeI, PstI, EcoRI, or XbaI)</li> | ||
+ | </ul> | ||
+ | <li>Set at 37°C for 3 hours</li> | ||
+ | <li>Heat inactivate at 80°C for 20 minute</li> | ||
+ | </ol> | ||
</div> | </div> | ||
<button class="protocol-accordion">Ligation</button> | <button class="protocol-accordion">Ligation</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <ol> | |
− | + | <li>Add the following to a PCR tube</li> | |
− | + | <ul> | |
− | + | <li>100ng of backbone</li> | |
− | + | <li>3:1 ratio of insert:backbone</li> | |
− | </ | + | <li> 2 μL 10X T4 DNA Ligase Buffer</li> |
+ | <li>1 μL T4 DNA Ligase</li> | ||
+ | <li> Add Cloning water to get total volume of 20uL</li> | ||
+ | </ul> | ||
+ | <li>Set at room temperature (~23°C) for 1-3 hours</li> | ||
+ | <li>Heat inactivate at 80°C for 20 minutes</li> | ||
+ | </ol> | ||
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</div> | </div> | ||
</div> | </div> | ||
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<h3> Measurement and Assays </h3> | <h3> Measurement and Assays </h3> | ||
− | <p> | + | <p style= " text-align: center;"> We used these protocols to test the effects of our plasmid constructs and quantify those results</p> |
</br> | </br> | ||
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− | <button class="protocol-accordion">ATP Extraction</button> | + | |
+ | <button class="protocol-accordion">ATP Induction/Extraction/Assay</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | <p> | + | <p style = "padding: 0px 0px 0px 50px;">Adapted from Thermo Fisher Scientific Molecular Probes® ATP Determination Kit</p> |
− | + | <a style = "padding: 30px 0px 30px 50px;" href = "https://static.igem.org/mediawiki/2016/6/62/T--WashU_StLouis--ATPassay.pdf">Link to Protocol</a> | |
− | + | ||
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− | </ | + | |
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</div> | </div> | ||
− | <button class="protocol-accordion">Electron Donor Extraction</button> | + | <button class="protocol-accordion">Electron Donor Induction/Extraction/Measurement and Biotin Assay</button> |
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <p style = "padding: 0px 0px 0px 50px;">Adapted from Vector Laboratories Quant*Tag Biotin Quantification Kit</p> | |
− | + | <a style = "padding: 30px 0px 30px 50px;" href = "https://static.igem.org/mediawiki/2016/4/49/T--WashU_StLouis--EDassay.pdf">Link to Protocol</a> | |
− | + | ||
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</div> | </div> | ||
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</div> | </div> | ||
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<h3> Miscellaneous </h3> | <h3> Miscellaneous </h3> | ||
− | <p> | + | <p style= " text-align: center;"> These protocols served a variety of important purposes </p> |
</br> | </br> | ||
<button class="protocol-accordion">Making Chemically Competent Cells</button> | <button class="protocol-accordion">Making Chemically Competent Cells</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <ol> | |
− | + | <li>Streak cells from frozen stock onto LB plate and incubate overnight at 37°C</li> | |
− | <li>Streak cells from frozen stock onto LB plate and incubate overnight at 37 C</li> | + | <li>Pick a colony and inoculate in LB (with antibiotic if applicable) and incubate overnight at 37°C</li> |
− | <li>Pick a colony and inoculate in LB (with antibiotic if applicable) and incubate overnight at 37 C</li> | + | <li>Dilute 1 mL of culture into 50 mL LBMgSO (with antibiotic if applicable) pre warmed to 37°C</li> |
− | <li>Dilute 1 mL of culture into 50 mL | + | <li>Grow culture in 37°C in a shaker until it reached an OD600 of 0.6 (2-5 hours depending on presence of antibiotic)</li> |
− | <li>Grow culture in 37 C in a shaker until it reached an OD600 of 0.6 (2-5 hours depending on presence of antibiotic)</li> | + | |
<li>Incubate cells for 20 minutes on ice</li> | <li>Incubate cells for 20 minutes on ice</li> | ||
<li>Transfer the cells to ice-cold sterile 50 mL tube</li> | <li>Transfer the cells to ice-cold sterile 50 mL tube</li> | ||
− | <li>Centrifuge for 10 minutes at 3000 rpm at 4 C and discard the supernatant</li> | + | <li>Centrifuge for 10 minutes at 3000 rpm at 4°C and discard the supernatant</li> |
− | <li>Gently resuspend the cells in 20 mL of ice-cold TFB1 | + | <li>Gently resuspend the cells in 20 mL of ice-cold TFB1 with chilled pipet tips</li> |
<li>Incubate cells on ice for 25 minutes</li> | <li>Incubate cells on ice for 25 minutes</li> | ||
<li>Repeat centrifuge, TFB1 resuspension, and 25 minutes on ice</li> | <li>Repeat centrifuge, TFB1 resuspension, and 25 minutes on ice</li> | ||
<li>Resuspend cells in 4 mL of ice-cold TFB2</li> | <li>Resuspend cells in 4 mL of ice-cold TFB2</li> | ||
<li>Aliquot 100 mL cells into prechilled 1.5 mL microcentrifuge tubes and freeze immediately in liquid nitrogen</li> | <li>Aliquot 100 mL cells into prechilled 1.5 mL microcentrifuge tubes and freeze immediately in liquid nitrogen</li> | ||
− | <li>Store at -80 C</li> | + | <li>Store at -80°C</li> |
− | </ | + | </ol> |
</div> | </div> | ||
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<button class="protocol-accordion">Making Antibiotic Plates</button> | <button class="protocol-accordion">Making Antibiotic Plates</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <ol> | |
− | + | ||
<li>Place 7.5 g Agar and 12.5 g Powdered LB Broth in a jar</li> | <li>Place 7.5 g Agar and 12.5 g Powdered LB Broth in a jar</li> | ||
− | <li>Fill to 500mL mark with | + | <li>Fill to 500mL mark with deionized water</li> |
<li>Add a magnetic stir bar and mix thoroughly using a stir plate</li> | <li>Add a magnetic stir bar and mix thoroughly using a stir plate</li> | ||
− | <li>Autoclave | + | <li>Autoclave</li> |
− | <li>Let cool, add 500 | + | <li>Let cool, add 500 μL of (each) antibiotic, and stir using stir plate</li> |
<li>In a biohood, Pipet 20-25 mL solution into labeled plates and allow to dry</li> | <li>In a biohood, Pipet 20-25 mL solution into labeled plates and allow to dry</li> | ||
− | <li>Store at 4 C</li> | + | <li>Store at 4°C</li> |
− | </ | + | </ol> |
</div> | </div> | ||
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<button class="protocol-accordion">Making Frozen Stock</button> | <button class="protocol-accordion">Making Frozen Stock</button> | ||
<div class="protocol-panel"> | <div class="protocol-panel"> | ||
− | + | <ol> | |
− | + | ||
<li>Inoculate cell culture</li> | <li>Inoculate cell culture</li> | ||
− | <li>Combine 500 | + | <li>Combine 500 μL of culture with 500 μL of 30% glycerol in a labeled cryo tube</li> |
− | <li>Invert to mix and store at -80 C</li> | + | <li>Invert to mix and store at -80°C</li> |
− | </ | + | </ol> |
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− | |||
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</div> | </div> | ||
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Latest revision as of 01:44, 20 October 2016
Experiments
Check out the protocols we used throughout our project
Cloning
We used these protocols to create plasmids with our genes of interest from genomic DNA, and transform those plasmids into E. coli
- Centrifuge incoulated cell culture for 15 minutes at 5000g
- Discard supernatant and resuspend in 1 mL of cloning water
- Transfer solution into bashing bead lysis/filtration tube
- Add 6 mL of fungal/bacterial DNA binding buffer
- Vortex for 5 minutes
- Spin down for 5 minutes at 5000g
- Transfer filter to a 50 mL tube
- Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g
- Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once
- Add 200 μL of fungal/bacterial DNA wash buffer to column, spin for 1 minute at 10,000g, and repeat 3 more times
- Transfer column to a 1.5 mL microcentrifuge tube and add 30 μL of cloning water
- Let sit for 4 minutes, then spin for 4 minutes at 10,000g
- Measure concentration of gDNA and label tube
- Add the following to a single PCR tube:
- 20-21.5 μL Cloning Water (depending on type of amplicon)
- 10 μL of Betaine
- 10 μL of 5x High-Fidelity Buffer
- 4 μL DMSO
- 2.5 μL 10 uM combined forward/reverse primers
- 1 μL DNTPs
- 0.5 μL of plasmid or 2 μL of gDNA
- 0.5 μL of Phusion Polymerase (kept on ice)
- Immediately run in a thermocycler for the following times/temperatures
- Initial Denaturation 98°C 30 sec
- 25-35 cycles
- 98°C 5-10 sec
- 45-72°C (at annealing temp) 10-30 sec
- 72°C 15-30 sec/(length of amplicon in kb)
- Final Extension 72°C 5-10 minutes
- Hold at 4°C
- Store at 4°C until use
- Measure 1 gram of Agarose per 100 mL TAE buffer (Tris base, acetic acid and EDTA)
- We generally made gels with 75 - 125 mL of TAE
- Microwave in a covered erlenmeyer flask for 120 seconds
- Add 1 μL of SYBR Safe or Ethidium Bromide per 20 mL of TAE
- Pour into gel casing
- Remove bubbles with a pipette tip
- Insert well comb
- Allow to set (~20 minutes)
- Remove well comb
- Place gel in electrophoresis apparatus with the wells by the cathode (black)
- Fill apparatus with TAE buffer so that the gel is completely submerged
- Load wells with 10-60 μL of 5 (PCR product): 1 (6x Loading Dye)
- Load 10 μL of ladder into first and last wells
- Run at 125-130 V for 25-30 minutes
(Adapted from Zymo Research Zymoclean™ Gel DNA Recovery Kit)
- Cut band from gel while minimizing time under UV light
- Place gel in a microcentrifuge tube with 3x volume of Agarose Dissolving Buffer
- Heat at 55°C until gel dissolves (~20 minutes)
- Add to gel purification filter placed inside a collection tube
- Centrifuge for 1 minute at 16,000 g and discard flow-through
- Add 200 μL of DNA wash buffer, centrifuge for 1 minute at 16,000 g, discard flow-through, and repeat once
- Centrifuge for 2 minutes at 16,000 g and discard flow-through
- Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water
- Wait for 4 minutes, then spin for 4 minutes at 16,000 g
- Measure concentrations and label
(Adapted from Zymo Research DNA Clean & Concentrator™-5 Kit)
- Add 50 μL DNA and 250 μL DNA Binding Buffer to a spin column in a collection tube
- Centrifuge for 30 seconds at Xg and discard flow-through
- Add 200 μL of DNA wash buffer, centrifuge for 30 seconds at 16,000g, discard flow-through, and repeat once
- Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water
- Wait for 4 minutes, then spin for 4 minutes at 16,000g
- Measure concentrations and label
- In a PCR tube, combine:
- 0.4 μL Cutsmart Buffer
- 0.4 μL DpnI
- plasmid backbone (either post PCR or gel extract)
- Incubate at 37°C for 1 hour
- DNA purification for post-DpnI mix to retrieve plasmid backbone
- In a PCR tube, combine:
- 100 ng of plasmid backbone
- Equimolar amount of each assembly piece
- 1.5 μL of restriction enzyme buffer
- 1 μL of ligase buffer
- 1 μL of Type IIs restriction enzyme
- 1 μL of T4 ligase
- Cloning water to equal a total of 15 μL
- Run in a thermocycler for the following times/temperatures:
- 50 cycles
- 3 minutes at 37°C
- 4 minutes at 16°C
- 1 cycle
- 5 minutes at 50°C
- 20 minutes at 80°C
- Hold at 4°C
- Transform into competent cells and/or store at -20°C
Electroporation
- Defrost competent cells on ice for 5 minutes
- Pipet 1.5 μL plasmid to the tube of competent cells
- Pipet the mixture into an electroporation cuvette
- Avoid touching the metal plate of the cuvette and introducing bubbles
- Shock
- Quickly add 500 μL of Lysogeny Broth (LB)
- Incubate in a culture tube for 1 hour at 37°C
- Pipet desired amount of solution onto a plate and spread evenly
- Allow solution to dry and incubate for 16-18 hours
- Wrap plate in parafilm and store at 4°C
Chemical Transdormation
- Thaw 100 μL of competent cells on ice
- Add 100 ng of plasmid to cells
- Incubate on ice for 20-30 minutes
- Heat shock the cells for 60 seconds at 42°C
- Return the cells to ice for 2 minutes
- Add 300 μL of LB or SOB medium
- Incubate in a culture tube for 1 hour at 37°C
- Pipet desired amount of solution onto a plate and spread evenly
- Allow solution to dry and incubate for 16-18 hours
- Wrap plate in parafilm and store at 4°C
Adapted from Zymo Research’s Zyppy Plasmid Miniprep kit
- Spin down cells for 15 minutes at 3200g
- Discard flow-through and resuspend in 600 μL of cloning water
- Transfer to a 1.5 mL microcentrifuge tube
- Add 100 μL of 6x Lysis Buffer and invert
- Add 300 μL of cold Neutralization Buffer and mix thoroughly
- Microcentrifuge for 4 minutes at 16,000g
- Pour supernatant into a miniprep filter placed in a collection tube
- Microcentrifuge for 1 minute at 16,000g and discard flow-through
- Add 200 μL of Endo-wash buffer
- Microcentrifuge for 30 seconds at 16,000g and discard flow-through
- Add 400 μL of Zyppy Wash buffer
- Microcentrifuge for 30 seconds at 16,000g and discard flow-through
- Microcentrifuge for 2 minutes at 16,000g and discard flow-through
- Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water
- Wait for 4 minutes, then spin for 4 minutes at 16,000g
- Measure concentrations and label
- Add 4 mL of Lysogeny broth (LB) and 4 μL of each needed antibiotic to a culture tube
- Add cells to the culture tube
- If using frozen stock, scrape a small amount of stock with a pipette tip
- If using a plate, carefully scrape a single colony with a pipette tip
- Grow at 37°C for 16-18 hours
BioBrick
We used these protocols to manipulate BioBricks provided in the iGEM distribution kit and to create our own BioBricks from experimentally tested genes
- Add the following to a PCR tube
- 29 μL of miniprepped plasmid
- 11 μL Cloning water
- 5 μL of NEB Buffer (depends on REs used)
- 5 μL total of Restriction Enzymes (SpeI, PstI, EcoRI, or XbaI)
- Set at 37°C for 3 hours
- Heat inactivate at 80°C for 20 minute
- Add the following to a PCR tube
- 100ng of backbone
- 3:1 ratio of insert:backbone
- 2 μL 10X T4 DNA Ligase Buffer
- 1 μL T4 DNA Ligase
- Add Cloning water to get total volume of 20uL
- Set at room temperature (~23°C) for 1-3 hours
- Heat inactivate at 80°C for 20 minutes
Measurement and Assays
We used these protocols to test the effects of our plasmid constructs and quantify those results
Adapted from Thermo Fisher Scientific Molecular Probes® ATP Determination Kit
Link to ProtocolAdapted from Vector Laboratories Quant*Tag Biotin Quantification Kit
Link to ProtocolMiscellaneous
These protocols served a variety of important purposes
- Streak cells from frozen stock onto LB plate and incubate overnight at 37°C
- Pick a colony and inoculate in LB (with antibiotic if applicable) and incubate overnight at 37°C
- Dilute 1 mL of culture into 50 mL LBMgSO (with antibiotic if applicable) pre warmed to 37°C
- Grow culture in 37°C in a shaker until it reached an OD600 of 0.6 (2-5 hours depending on presence of antibiotic)
- Incubate cells for 20 minutes on ice
- Transfer the cells to ice-cold sterile 50 mL tube
- Centrifuge for 10 minutes at 3000 rpm at 4°C and discard the supernatant
- Gently resuspend the cells in 20 mL of ice-cold TFB1 with chilled pipet tips
- Incubate cells on ice for 25 minutes
- Repeat centrifuge, TFB1 resuspension, and 25 minutes on ice
- Resuspend cells in 4 mL of ice-cold TFB2
- Aliquot 100 mL cells into prechilled 1.5 mL microcentrifuge tubes and freeze immediately in liquid nitrogen
- Store at -80°C
- Place 7.5 g Agar and 12.5 g Powdered LB Broth in a jar
- Fill to 500mL mark with deionized water
- Add a magnetic stir bar and mix thoroughly using a stir plate
- Autoclave
- Let cool, add 500 μL of (each) antibiotic, and stir using stir plate
- In a biohood, Pipet 20-25 mL solution into labeled plates and allow to dry
- Store at 4°C
- Inoculate cell culture
- Combine 500 μL of culture with 500 μL of 30% glycerol in a labeled cryo tube
- Invert to mix and store at -80°C