Team:WashU StLouis/Protocol
From 2014.igem.org
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<a href="#7">Starting Cultures</a> | <a href="#7">Starting Cultures</a> | ||
<a href="#8">Miniprep</a> | <a href="#8">Miniprep</a> | ||
+ | <a href="#9">Light Induction</a> | ||
<br> | <br> | ||
- | <a href="# | + | <center><b>Rebstock Group</b></center> |
+ | <a href="#10">Electroporation</a> | ||
+ | <a href="#11">Innoculation</a> | ||
+ | <a href="#12">10x M9 Stock</a> | ||
+ | <a href="#13">100x Ferric Citrate Soln</a> | ||
+ | <a href="#14">1x K.pneumoniae Medium</a> | ||
+ | <a href="#15">Acetylene Reduction Assay</a> | ||
+ | <a href="#16">Gel Electrophoresis</a> | ||
+ | <a href="#17">CPEC</a> | ||
+ | |||
</div> | </div> | ||
Line 34: | Line 44: | ||
<table id="menu" width=100%" cellspacing="0" height="500px"> | <table id="menu" width=100%" cellspacing="0" height="500px"> | ||
<tr> | <tr> | ||
- | <td width="600px" align="center" bgColor="# | + | <td width="600px" align="center" bgColor="#FFF" > |
<table id="general"> | <table id="general"> | ||
- | <tr> <td> | + | <tr> <td><body> |
- | <center><h1> | + | <div style="text-align: center;"> |
- | + | <h1><span style="font-weight: bold;">Brauer Group Protocols</span></h1> | |
- | < | + | <span style="font-weight: bold;"></span></div> |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | < | + | |
<a name="1"></a> | <a name="1"></a> | ||
- | < | + | <span style="font-weight: bold;">Polymerase Chain Reaction</span><img |
- | + | style="width: 200px; height: 267px;" alt="Thermal Cycler" | |
- | + | src="https://static.igem.org/mediawiki/2014/b/ba/WashU_Thermal_Cycler.jpg" | |
- | + | align="right"><br> | |
- | + | <br> | |
- | < | + | Phusion HF Polymerase Protocol<br> |
- | + | <div style="margin-left: 40px;">Per tube, add in this | |
- | < | + | order: |
- | + | | |
- | <table border="1" | + | |
+ | <br> | ||
+ | </div> | ||
+ | <table style="width: 197px; height: 320px; margin-left: 40px;" | ||
+ | border="1" cellpadding="1" cellspacing="0"> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td | + | <td style="vertical-align: top; text-align: center;"> Cloning |
- | <td valign="top" | + | water </td> |
+ | <td style="text-align: center;" valign="top"> 21.5 µl </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 5x HF buffer </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 10 µl </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 5M Butane </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 10 µl </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> DMSO </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 4 µl </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 10µM Primers (f+r) </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 2.5 µl </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 10µM dNTPs </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 1.0 µl </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> Template DNA </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 0.5 µl </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> Phusion Polymerase </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 0.5 µ </td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | + | PCR Reaction in Thermal Cycler: | |
- | + | <table style="height: 40px; margin-left: 40px;" border="1" | |
- | + | cellpadding="1" cellspacing="0"> | |
- | <table border="1" | + | |
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 98 °C </td> |
- | <td valign="top" | + | <td valign="top"> 30 seconds </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 30 Cycles </td> |
- | <td valign="top"> <table | + | <td valign="top"> |
+ | <table style="width: 316px; height: 187px;" border="1" | ||
+ | cellpadding="5" cellspacing="0"> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 98 °C </td> |
- | <td valign="top" | + | <td valign="top"> 20 seconds </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> Annealing Temperature (NEB TM Calculator) |
- | <td valign="top" | + | </td> |
+ | <td valign="top"> 20 seconds </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 72 °C </td> |
- | <td valign="top" | + | <td valign="top"> Extension Time (20 seconds/kb) </td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | |||
</td> | </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 72 °C </td> |
- | <td valign="top" | + | <td valign="top"> 5 minutes </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 4 °C </td> |
- | <td valign="top" | + | <td valign="top"> Forever </td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | |||
<br> | <br> | ||
- | + | <br> | |
<a name="2"></a> | <a name="2"></a> | ||
- | + | Colony PCR<br> | |
- | + | <div style="margin-left: 40px;">1. Prepare 10 µl of cloning water per | |
- | < | + | colony to be picked in PCR tubes.<br> |
- | + | 2. Prepare culture tube filled with 4mL LB, 4µL resistance<br> | |
- | + | 3. Pick colony and resuspend in cloning water by pipetting up and down<br> | |
- | + | 4. Next, drop the tip inside the culture tube, or dot onto pre-numbered | |
- | + | LB plate<br> | |
- | + | 5. Heat water with colonies for 10 minutes in 98°C; use as “template | |
- | + | DNA”<br> | |
- | + | 6. Put culture tubes in 37°C shaker to incubate (overnight) while PCR | |
- | + | is running.<br> | |
- | + | 7. Go TAQ PCR mix<br> | |
- | + | </div> | |
- | + | <table style="height: 246px; width: 255px; margin-left: 40px;" | |
- | + | border="1" cellpadding="5" cellspacing="0"> | |
- | + | ||
- | <table border="1" | + | |
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> Cloning Water </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 35µl </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> Vortexed 5x Green |
- | <td valign="top" | + | Taq Buffer </td> |
+ | <td style="text-align: center;" valign="top"> 10µl </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 10µM Sequencing |
- | <td valign="top" | + | Primer </td> |
+ | <td style="text-align: center;" valign="top"> 2.5µl </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 10µM dNTPs </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 1.0µl </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> Template DNA |
- | <td valign="top" | + | (heated above) </td> |
+ | <td style="text-align: center;" valign="top"> 1.0µl </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> GoTaq Polymerase </td> |
- | <td valign="top" | + | <td style="text-align: center;" valign="top"> 0.5µl </td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | + | <div style="margin-left: 40px;">8. TAQ PCR Protocol | |
- | < | + | </div> |
- | + | <table style="width: 392px; height: 136px; margin-left: 40px;" | |
- | <table | + | border="1" cellpadding="5" cellspacing="0"> |
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 95 °C </td> |
- | <td valign="top" | + | <td valign="top"> 2 minutes </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 35 Cycles </td> |
- | <td valign="top"> <table | + | <td valign="top"> |
+ | <table style="width: 303px; height: 214px;" border="1" | ||
+ | cellpadding="5" cellspacing="0"> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 95 °C </td> |
- | <td valign="top" | + | <td valign="top"> 1 minute </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> Annealing Temperature (NEB TM Calculator) |
- | <td valign="top" | + | </td> |
+ | <td valign="top"> 45 seconds </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 72 °C </td> |
- | <td valign="top" | + | <td valign="top"> Extension Time (20 seconds/kb) </td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | |||
</td> | </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 72 °C </td> |
- | <td valign="top" | + | <td valign="top"> 5 minutes </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 4 °C </td> |
- | <td valign="top" | + | <td valign="top"> Forever </td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | + | <div style="margin-left: 40px;"><br> | |
- | < | + | 9. Run Gel Visualization<br> |
- | + | 10. Add 15µl PCR product (no need 6x loading dye)<br> | |
- | + | 11. Freeze & Miniprep cultures that have desired bands<br> | |
- | + | </div> | |
- | + | ||
- | + | ||
<br> | <br> | ||
- | + | <span style="font-weight: bold;">Gradient PCR</span><br> | |
- | < | + | <ul> |
- | + | <li>Annealing Temperature: 5 °C below to 5 °C above.</li> | |
- | < | + | <li>Example: 62°C = 57°C -> 67°C</li> |
- | + | <li>Increase Annealing Time to 30 seconds</li> | |
- | < | + | </ul> |
- | + | <br style="font-weight: bold;"> | |
- | < | + | <a name="3"></a><span style="font-weight: bold;">Making a Gel for Gel |
- | + | Electrophoresis</span><img style="width: 200px; height: 267px;" | |
+ | alt="Making a gel" | ||
+ | src="https://static.igem.org/mediawiki/2014/7/72/WashU_Making_Gel.jpg" | ||
+ | align="right"><br> | ||
+ | <ol> | ||
+ | <li>0.7g of Agarose</li> | ||
+ | <li>Add 100 ml 1x TAE buffer into an Erlenmeyer flask</li> | ||
+ | <li>Microwave for 2 minutes</li> | ||
+ | <li>Cool down under tap water for 20 seconds</li> | ||
+ | <li>Add 5 µl Sybrsafe</li> | ||
+ | <li>Pour the gel into mold with comb. Let cool for 30 minutes.</li> | ||
+ | <li>Remove the comb.</li> | ||
+ | <li>Transfer tray into an electrophoresis chamber, wells facing black | ||
+ | side (anode) of chamber</li> | ||
+ | <li>Fill chamber with 1x TAE until TAE just covers the gel</li> | ||
+ | </ol> | ||
+ | <span style="font-weight: bold;"> <br> | ||
+ | Gel visualization/extraction</span><br> | ||
<br> | <br> | ||
- | < | + | <div style="margin-left: 40px;">1. Prepare samples<br> |
- | + | </div> | |
- | + | <table | |
- | + | style="width: 292px; height: 40px; text-align: left; margin-left: 40px;" | |
- | + | border="1" cellpadding="1" cellspacing="0"> | |
- | + | ||
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- | <br> | + | |
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- | < | + | |
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td | + | <td style="vertical-align: top; text-align: left;"> Visualization |
- | <td valign="top" | + | </td> |
+ | <td style="text-align: left;" valign="top"> Extraction </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 10µl PCR Product </td> |
- | <td valign="top" | + | <td valign="top"> 50µl PCR Product </td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top"> 2µl 6x Loading Dye </td> |
- | <td valign="top" | + | <td valign="top"> 10µl 6x Loading Dye </td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | + | <div style="margin-left: 40px;"><br> | |
- | < | + | 2. Add DNA ladder Standard into first well.<br> |
- | + | 3. Pipet prepared samples into rest of wells.<br> | |
- | + | 4. Seal electrophoresis chamber with chamber cap and plug cables in VWR | |
- | + | power unit.<br> | |
- | + | 5. Set 125V, Run for 30 minutes (or longer) depending on length of | |
- | + | sample expected.<br> | |
- | + | 6. Remove Gel tray once finished and visualize using trans-illuminator.<br> | |
- | + | 7. Extract if necessary into a preweighed 1.5 ml microcentrifuge tube.<br> | |
- | + | </div> | |
- | + | ||
- | + | ||
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- | </ | + | |
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<br> | <br> | ||
- | + | <span style="font-weight: bold;">Gel purification</span><img | |
- | < | + | style="width: 200px; height: 267px;" alt="Gel purification kit" |
- | + | src="https://static.igem.org/mediawiki/2014/7/70/WashU_Gel_Purify.jpg" | |
- | + | align="right"><br> | |
- | + | ||
- | + | ||
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- | + | ||
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<br> | <br> | ||
- | < | + | <div style="margin-left: 40px;">Using Zymoclean Gel DNA Recovery Kit<br> |
- | + | 1. Add 3 volumes (µl) for each volume of agarose (mg) excised from the | |
- | + | gel.<br> | |
- | + | 2. Incubate at 55°C for 10 minutes until gel is dissolved<br> | |
- | + | 3. Pipette into a Zymo-Spin Column & Collection Tube<br> | |
- | + | 4. Centrifuge (at 16000 x g)for 1 minute, and discard the flow-through.<br> | |
- | + | 5. Add 200µl DNA wash Buffer and centrifuge for 30 seconds.<br> | |
- | + | 6. Add another 200µl DNA wash buffer and centrifuge for a minute.<br> | |
- | + | 7. Place the spin column into a new sterile microcentrifuge tube.<br> | |
- | + | 8. Add up to 30µl of Cloning Water directly to the column matrix.<br> | |
- | + | 9. Let it sit for 4 minutes, and centrifuge for 4 minutes.<br> | |
- | + | </div> | |
- | + | <br> | |
- | + | <span style="font-weight: bold;">Treating with DPNI</span><br> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
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<br> | <br> | ||
- | <a name=" | + | <div style="margin-left: 40px;">1. Add to a PCR rxn mix after PCR.<br> |
- | < | + | 2. 1µl DPNI per 50 µl rxn mix.<br> |
- | + | 3. Let it run 37°C for 1 hour.<br> | |
- | < | + | </div> |
- | + | <br style="font-weight: bold;"> | |
- | + | <a name="4"></a><span style="font-weight: bold;">DNA Purification</span><img | |
- | + | style="width: 270px; height: 203px;" alt="DNA Purification kit" | |
- | + | src="https://static.igem.org/mediawiki/2014/0/07/WashU_PCR_Purify.jpg" | |
- | + | align="right"><br> | |
- | + | <br> | |
- | + | <div style="margin-left: 40px;">Using Zymoclean DNA Clean & | |
- | + | Concentrator<br> | |
- | + | 1. Add 5 volumes of DNA binding Buffer to PCR product or Short DNA | |
- | + | fragments.<br> | |
- | + | 2. Vortex Tube.<br> | |
- | + | 3. Load mixture into Zymo-Spin Column and Collection Tube<br> | |
- | + | 4. Centrifuge (16000 x g) for 30 seconds. Discard flow-through.<br> | |
- | + | 5. Add 200µl of DNA Wash Buffer and centrifuge for 30 seconds.<br> | |
- | + | 6. Add another 200µl of DNA Wash Buffer and centrifuge for 2 minutes.<br> | |
- | + | 7. Place the spin column into a new sterile microcentrifuge tube.<br> | |
- | + | 8. Add up to 30µl of Cloning Water directly to the column matrix.<br> | |
- | <table border="1" | + | 9. Let it sit for 4 minutes, and centrifuge for 4 minutes.<br> |
+ | </div> | ||
+ | <br> | ||
+ | <a name="5"></a><span style="font-weight: bold;">Golden Gate | ||
+ | Digestion/Ligation</span><br> | ||
+ | <br> | ||
+ | <div style="margin-left: 40px;">1. Design Primers accordingly, taking | ||
+ | into account Type IIs Restriction | ||
+ | Enzyme site.<br> | ||
+ | 2. Add purified pieces you want to ligate based on concentration | ||
+ | results (100ng / x ng/µl) into a PCR tube.<br> | ||
+ | 3. Add cloning water to the mix up to 10.5µl total.<br> | ||
+ | 4. Add 1.5µl Cutsmart Buffer to the mix.<br> | ||
+ | 5. Vortex ligase buffer. Add 1.0µl to the mix.<br> | ||
+ | 6. Add 1µl of Type IIs restriction enzyme (we usually use sapI)<br> | ||
+ | 7. Add 1µl of T4 Ligase to the mix.<br> | ||
+ | 8. Total should be 15µl in the tube. If volume of pieces you want to | ||
+ | ligate together add up to greater than 10.5µl, scale up the mix | ||
+ | proportionally.<br> | ||
+ | Reaction assembly<br> | ||
+ | </div> | ||
+ | <table style="width: 148px; height: 232px; margin-left: 40px;" | ||
+ | border="1" cellpadding="1" cellspacing="0"> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top">50 x</td> |
- | <td valign="top"> <table | + | <td valign="top"> |
+ | <table style="width: 95px; height: 104px;" border="1" | ||
+ | cellpadding="5" cellspacing="0"> | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top">37°C</td> |
- | <td valign="top" | + | <td valign="top">3 min</td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top">16°C</td> |
- | <td valign="top" | + | <td valign="top">4 min</td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | |||
</td> | </td> | ||
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top">50°C</td> |
- | <td valign="top" | + | <td valign="top">5 min</td> |
</tr> | </tr> | ||
- | |||
<tr> | <tr> | ||
- | <td valign="top" | + | <td valign="top">80°C</td> |
- | <td valign="top" | + | <td valign="top">5 min</td> |
</tr> | </tr> | ||
- | |||
</tbody> | </tbody> | ||
</table> | </table> | ||
- | |||
<br> | <br> | ||
- | + | <span style="font-weight: bold;">Restriction Enzyme Digestion</span><br> | |
- | < | + | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<br> | <br> | ||
- | + | <div style="margin-left: 40px;">1. Follow NEB protocols.<br> | |
- | + | 2. Find appropriate buffer if doing double digests.<br> | |
- | + | 3. Heat inactivate at the end.<br> | |
- | < | + | 4. If doing sequential digests, heat inactivate in between each step.<br> |
- | + | </div> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<br> | <br> | ||
- | < | + | <span style="font-weight: bold;">Blunt End Ligation</span><br> |
- | < | + | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<br> | <br> | ||
- | < | + | <div style="margin-left: 40px;">1. Add 18µl of purified PCR product |
- | < | + | into a PCR tube.<br> |
- | + | 2. Add 2.25µl of 10x T4 Ligase Buffer<br> | |
- | < | + | 3. Add 1.125µl of T4 PolyNucleotide Kinase.<br> |
- | + | 4. Put in thermocycler for 37°C for 45 minutes.<br> | |
- | + | 5. Add 1.125µl T4 DNA Ligase<br> | |
- | + | 6. Leave at room temperature for at least 1 hour.<br> | |
- | < | + | 7. Heat inactivate whatever you are not using at 65°C for 20 minutes.<br> |
- | + | </div> | |
- | < | + | <br style="font-weight: bold;"> |
- | + | <a name="6"></a><span style="font-weight: bold;">Transformation | |
- | < | + | (Electroporation)</span><br> |
- | + | ||
- | < | + | |
- | + | ||
<br> | <br> | ||
- | + | <div style="margin-left: 40px;">1. Put electroporation cuvette on ice | |
- | < | + | and thaw electro-competent cells |
- | + | for 5 minutes.<br> | |
- | < | + | 2. Prepare 500µl LB and a labeled culture tube.<br> |
- | + | 3. Add up to 5µl of DNA directly to the competent cells. (usually use | |
- | < | + | 2µl)<br> |
- | + | 4. Transfer mix into cuvette, ensuring no bubbles and mix touching both | |
- | < | + | sides.<br> |
- | + | 5. Wipe down cuvette to remove moisture.<br> | |
- | < | + | 6. Place in electroporation machine and start.<br> |
- | + | 7. If arc occurs, try with smaller amounts of DNA.<br> | |
- | + | 8. If successful, immediately add the LB directly into the cuvette.<br> | |
- | + | 9. Transfer LB + transformed cells into the culture tube.<br> | |
- | + | 10. Record time and incubate in the 37°C shaker for 1 hour.<br> | |
- | + | </div> | |
<br> | <br> | ||
- | + | <a name="7"></a><span style="font-weight: bold;">Plating</span><br> | |
- | < | + | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | < | + | |
- | + | ||
<br> | <br> | ||
- | <a name="8"></a> | + | <div style="margin-left: 40px;">1. Prepare two appropriate antibiotic |
- | < | + | resistance “100µl” and “Rest”<img style="width: 200px; height: 329px;" |
- | + | alt="Plating" | |
- | < | + | src="https://static.igem.org/mediawiki/2014/e/e3/WashU_Week9.jpg" |
- | + | align="right"><br> | |
- | < | + | 2. Add 100µl of transformed culture (after the 1 hour incubation) onto |
- | + | the 100µl plate.<br> | |
- | + | 3. Use a sterile spreader to streak the liquid culture onto the plate | |
- | + | in the order on the left:<br> | |
- | + | 4. On the “Rest” Plate, pour the rest of the liquid culture and spread | |
- | + | evenly on the plate.<br> | |
- | + | 5. Flip and put in the 37°C incubator overnight (no longer than 20 | |
- | + | hours).<br> | |
- | + | 6. Wrap your plate after sufficiently grown and store in the 4°C fridge | |
- | + | for up to 45 days.<br> | |
- | + | </div> | |
- | + | <br style="font-weight: bold;"> | |
- | + | <span style="font-weight: bold;">Liquid Cultures</span><br> | |
- | + | <br> | |
- | + | <div style="margin-left: 40px;">1. Determine volume of culture to grow | |
- | + | in a culture tube. We usually | |
- | + | use 4ml of LB.<br> | |
- | + | 2. In the biosafety hood, add 4ml of LB, and 4µl of each appropriate | |
- | + | resistance(s).<br> | |
- | + | 3. Use a tip to pick up a single colony from the plate.<br> | |
- | + | 4. Drop the tip into the media.<br> | |
- | + | 5. Cap the culture tube to the first stop.<br> | |
- | + | 6. Incubate in 37°C shaker at 250rpm for up to 16 hours.<br> | |
- | + | </div> | |
- | + | <br> | |
- | + | <a name="8"></a><span style="font-weight: bold;">Freeze Cultures</span><br> | |
- | </ | + | <br> |
- | </ | + | <div style="margin-left: 40px;">1. Add 500µl of 30% glycerol into a |
- | </ | + | cryo-freeze tube.<br> |
- | + | 2. Add 500µl of overnight culture.<br> | |
- | < | + | 3. Store in -80°C.<br> |
- | < | + | </div> |
- | <center> <h1> Rebstock Group Protocols </h1> </ | + | <br style="font-weight: bold;"> |
- | <a name="2"></a> | + | <span style="font-weight: bold;">Plasmid Miniprep</span><br> |
+ | <br> | ||
+ | <div style="margin-left: 40px;">Using Zyppy Plasmid Miniprep Kit<img | ||
+ | style="width: 270px; height: 203px;" alt="Miniprep" | ||
+ | src="https://static.igem.org/mediawiki/2014/0/0e/WashU_Miniprep.jpg" | ||
+ | align="right"><br> | ||
+ | 1.Centrifuge overnight culture for 10 minutes at 5000 x g<br> | ||
+ | 2. Discard supernatant. Resuspend with 550µl LB<br> | ||
+ | 3. Transfer culture to 1.5 ml microcentrifuge tube.<br> | ||
+ | 4. Add 100 µl of 7X Lysis Buffer (Blue). Shake well.<br> | ||
+ | 5. Within 2 minutes, add 350 µl of cold Neutralization Buffer (Yellow). | ||
+ | Shake well.<br> | ||
+ | 6. Centrifuge at 16000 x g for 4 minutes.<br> | ||
+ | 7. Transfer supernatant into a Zymo-Spin Column and Collection Tube.<br> | ||
+ | 8. Centrifuge at 16000 x g for 30 seconds. Discard flow-through.<br> | ||
+ | 9. Add 200µl Endo-Wash Buffer. Centrifuge at 16000 x g for 30 seconds.<br> | ||
+ | 10. Add 400µl Zyppy Wash Buffer. Centrifuge at 16000 x g for 2 minutes.<br> | ||
+ | 11. Transfer column into new labeled microcentrifuge tube.<br> | ||
+ | 12. Add 30µl of cloning water directly to column matrix. Let sit for 4 | ||
+ | minutes.<br> | ||
+ | 13. Centrifuge at 16000 x g for 4 minutes to elute.<span | ||
+ | style="font-weight: bold;"></span><br> | ||
+ | <span style="font-weight: bold;"></span></div> | ||
+ | <span style="font-weight: bold;"></span><br> | ||
+ | <a name="9"></a><span style="font-weight: bold;">Light Induction | ||
+ | Experiment</span><br> | ||
+ | <br> | ||
+ | <div style="margin-left: 40px;">1. Co-transform chromophore plasmid | ||
+ | with light sensor (2µl of each miniprepped product).<img | ||
+ | style="width: 200px; height: 270px;" alt="Light Induction Expt" | ||
+ | src="https://static.igem.org/mediawiki/2014/1/14/WashU_Light_induction_expt.jpg" | ||
+ | align="right"><br> | ||
+ | 2. Plate and pick colonies.<br> | ||
+ | 3. Start liquid cultures with appropriate antibiotic resistance for | ||
+ | light sensor(s) and appropriate controls.<br> | ||
+ | 4. Wrap tubes that you want to use in dark with foil carefully.<br> | ||
+ | 5. Freeze experimental stock for future use.<br> | ||
+ | 6. Measure OD600<br> | ||
+ | 7. Dilute to OD 0.1<br> | ||
+ | 8. Grow for 2 hours in 37°C shaker.<br> | ||
+ | 9. Transfer into deep well plates and induce with No and Max ATC in | ||
+ | both Light and Dark conditions (2 controls, light and ATC).<br> | ||
+ | 10. Grow for 6-8 hours in a lighted growth chamber (wrap plates that | ||
+ | will be in the dark).<br> | ||
+ | 11. Spin down colonies at 3000x g for 15 minutes.<br> | ||
+ | 12. Resuspend with 200µl 1 x PBS.<br> | ||
+ | 13. Measure Fluorescence/Absorbtion in 96 well plates. <br> | ||
+ | 14. For EYFP we used | ||
+ | Excitation and Emission values of 485nm and 528nm respectively.<br> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;"></span></div> | ||
+ | <span style="font-weight: bold;"></span><br> | ||
+ | <div style="text-align: center;"> | ||
+ | <h1>Rebstock Group Protocols</h1> | ||
+ | </div> | ||
+ | <a name="10"></a><span style="font-weight: bold;">Electroporation | ||
+ | Protocol<br> | ||
+ | <br> | ||
+ | </span> | ||
+ | <ol> | ||
+ | <li>Take 1 mL from tube culture and transfer it into 50 mL of LB | ||
+ | broth in a flask. Let culture for 2 hrs in the 37°C room.</li> | ||
+ | <li>Pipet 150 μL of each flask into a well in a 96 well plate and | ||
+ | analyze the optical density. May proceed when OD600 is between 0.1 and | ||
+ | 0.15</li> | ||
+ | <li>Chill the flasks for 15 min. and shake/swirl every 5 min.</li> | ||
+ | <li>Empty the flasks into centrifuge tubes</li> | ||
+ | <li>Centrifuge for 5 mins at 4000 rpm. </li> | ||
+ | <li>Extract supernatant with vacuum and fill with autoclaved water to | ||
+ | between 15-20 mL--just make sure they are all even. </li> | ||
+ | <li>Repeat step 5</li> | ||
+ | <li>Repeat step 6</li> | ||
+ | <li>Repeat step 5</li> | ||
+ | <li>Add 200 μL of 10% glycerol into each tube</li> | ||
+ | <li>Pipet 50 μL of each into a cuvette</li> | ||
+ | <li>Pipet 4 μL of plasmid suspension into the cuvette and ice for 10 | ||
+ | min. </li> | ||
+ | <li>Transfer to electroporation cuvette--be sure not to touch the | ||
+ | metal--and keep on ice</li> | ||
+ | <li>Perform electroporation under the second E. coli setting: 2500 V | ||
+ | with a 0.2 cm cuvette</li> | ||
+ | <li>Transfer 1 mL of LB to each electroporation cuvette.</li> | ||
+ | <li>Transfer all of the contents of the cuvettes into tubes. Grow for | ||
+ | 1 hr at 37°C</li> | ||
+ | <li>Transfer the contents of the tubes into centrifugable cuvettes</li> | ||
+ | <li>Plate varying amounts of the cell suspension. **For large volumes | ||
+ | (>500 μL) spin down the desired volume in a small centrifuge, then | ||
+ | remove most of the supernatant so that the volume is ~150 μL. Resuspend | ||
+ | the cells and plate the remaining suspension.</li> | ||
+ | <li>Make sure the plates are dry before storing them at 37°C | ||
+ | overnight or until discernible colonies form.</li> | ||
+ | </ol> | ||
+ | <br style="font-weight: bold;"> | ||
+ | <a name="11"></a><span style="font-weight: bold;">Inoculation of | ||
+ | Transformed E. coli | ||
+ | from Plates into Tubes Protocol </span><img | ||
+ | style="width: 250px; height: 250px;" alt="innoculating media" | ||
+ | src="https://static.igem.org/mediawiki/2014/f/f5/WashU_Culturing_Ecoli.jpg" | ||
+ | align="right"><br> | ||
+ | <ol> | ||
+ | <li>Prepare all labels before hand--be sure to include culture | ||
+ | identifiers, the date, and your initials</li> | ||
+ | <li>Light a bunsen burner and perform all steps using sterile | ||
+ | technique</li> | ||
+ | <li>Pipet 4 mL of LB into each tube using a volumetric pipet</li> | ||
+ | <li>Using a 10 μL pipettman, add 4 μL of the appropriate antibiotic | ||
+ | to each tube</li> | ||
+ | <li>Now put a tip on your 10 μL pipettman and scrape a single colony | ||
+ | from your plate</li> | ||
+ | <li>Eject the tip into the tube and cap the tube</li> | ||
+ | <li>Place all tubes on the shaker table in the 37°C room</li> | ||
+ | </ol> | ||
+ | <br> | ||
+ | <a name="12"></a><span style="font-weight: bold;">Recipe for 10X M9 | ||
+ | Stock Solution for | ||
+ | Nitrogenase Activity Assay (100mL): </span> <br> | ||
+ | <br> | ||
+ | Reagents: | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li>0.026 g CaCl2·H2O, </li> | ||
+ | <li>0.030 g MgSO4,</li> | ||
+ | <li>10.4 g Na2HPO4, </li> | ||
+ | <li>3.4 g KH2PO4, </li> | ||
+ | <li>4 g glucose </li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | Procedure: | ||
+ | <ol> | ||
+ | <li>Fill a clean 100 mL beaker with ~70 mL of water</li> | ||
+ | <li>Add a magnetic stir bar and place on stir plate, stirring | ||
+ | moderately</li> | ||
+ | <li>Add the appropriate amount of reagent listed above IN THE ORDER | ||
+ | listed above</li> | ||
+ | <li>Increase or decrease the speed of the stir plate until all | ||
+ | particles are dissolved</li> | ||
+ | <li>Once the solution is clear and colorless, remove the stir bar and | ||
+ | pour the contents of the beaker into a 100 mL | ||
+ | graduated cylinder</li> | ||
+ | <li>Fill with water to the 100 mL mark</li> | ||
+ | <li>Pour the solution back into the original beaker</li> | ||
+ | <li>In a hood with a flame, filter the solution with a sterile filter | ||
+ | into an autoclaved bottle</li> | ||
+ | <li>Label and store for future use </li> | ||
+ | </ol> | ||
+ | <br> | ||
+ | <span style="font-weight: bold;">Recipe for 1000X M9 Supplemental Stock | ||
+ | Solution (100 mL) </span><img style="width: 250px; height: 250px;" | ||
+ | alt="Storing solutions" | ||
+ | src="https://static.igem.org/mediawiki/2014/d/d9/WashU_storing_solutions.JPG" | ||
+ | align="right"> | ||
+ | <br> | ||
+ | Reagents: | ||
+ | <br> | ||
+ | <ul style="margin-left: 40px;"> | ||
+ | <li>0.3 g MnSO4, | ||
+ | </li> | ||
+ | <li>7.6 g Na2MoO4*2H2O, </li> | ||
+ | <li>0.010 g p-aminobenzoic acid, </li> | ||
+ | <li>0.005 g biotin</li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | </ul> | ||
+ | Procedure: | ||
+ | <ol> | ||
+ | <li>Fill a 100 mL beaker with ~70 mL of water</li> | ||
+ | <li>Add a magnetic stir bar and place on stir plate, stirring | ||
+ | moderately</li> | ||
+ | <li>Add the appropriate amount of reagent listed above</li> | ||
+ | <li>Increase or decrease the speed of the stir plate until all | ||
+ | particles are dissolved</li> | ||
+ | <li>Remove the stir bar and pour the contents of the beaker into a | ||
+ | 100 mL graduated cylinder</li> | ||
+ | <li>Fill with water to the 100 mL mark </li> | ||
+ | <li>Pour the solution back into the original beaker</li> | ||
+ | <li>In a hood with a flame, filter the soution with a sterile filter | ||
+ | into an autoclaved bottle</li> | ||
+ | <li>Label and store for future use</li> | ||
+ | </ol> | ||
+ | <br style="font-weight: bold;"> | ||
+ | <a name="13"></a><span style="font-weight: bold;">Recipe for 100X | ||
+ | Ferric Citrate Stock | ||
+ | Solution (100 mL)</span><br> | ||
+ | <br> | ||
+ | Reagent: | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li>0.36 g Ferric citrate</li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | Procedure: | ||
+ | <ol> | ||
+ | <li>Fill a 100 mL beaker with ~70 mL of water</li> | ||
+ | <li>Add a magnetic stir bar and place on a stir plate, stirring | ||
+ | moderately</li> | ||
+ | <li>Add the appropriate amount of reagent listed above</li> | ||
+ | <li>Increase or decrease the speed of the stir plate until the | ||
+ | surface of the particles have dissolved--not all of the rusty-colored | ||
+ | particles will dissolve</li> | ||
+ | <li>Transfer the suspension into an autoclaveable bottle</li> | ||
+ | <li>Autoclave</li> | ||
+ | <li>Once removed from the autoclave, the particles should be | ||
+ | dissolved and the solution should be a yellow-brown color. </li> | ||
+ | <li>Label and store for future use</li> | ||
+ | </ol> | ||
+ | <br> | ||
+ | <a name="14"></a><span style="font-weight: bold;">Recipe for Other 1X | ||
+ | K. pneumoniae | ||
+ | Medium (100 mL)</span><img style="width: 250px;" alt="image" | ||
+ | src="https://static.igem.org/mediawiki/2014/b/b6/WashU_Solution_pic_2.tiff" | ||
+ | align="right"><br> | ||
+ | <br> | ||
+ | Mix the following to make 100 mL of solution: | ||
+ | <ul> | ||
+ | <ul> | ||
+ | <li>0.01g CaCl22H2O</li> | ||
+ | <li>0.025g MgSO47H2O</li> | ||
+ | <li>2.5 g Na2HPO4</li> | ||
+ | <li>0.3g KH2PO4</li> | ||
+ | <li>0.1g NaCl</li> | ||
+ | <li>0.00029 g FeCl3</li> | ||
+ | <li>0.000025 g Na2MoO42H2O</li> | ||
+ | <li>2.0 g sucrose </li> | ||
+ | <li>0.6 mL (per liter) of 22% (wt/vol) NH4Ac</li> | ||
+ | </ul> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <a name="15"></a><span style="font-weight: bold;">Preparation for | ||
+ | Acetylene Reduction | ||
+ | Assay </span><img style="width: 200px; height: 200px;" alt="GC machine" | ||
+ | src="https://static.igem.org/mediawiki/2014/2/28/WashU_GC_Machine.jpg" | ||
+ | align="right"><br> | ||
+ | <ol> | ||
+ | <li>Grow engineered E. coli strains at 37°C overnight in 4 mL of LB | ||
+ | broth with antibiotics</li> | ||
+ | <li>Collect cultures by centrifugation and wash with sterilized water | ||
+ | three times </li> | ||
+ | <li>Resuspend cells in 5 mL of nitrogen-deficient medium with 10 mM | ||
+ | glutamate as a nitrogen source in a sealed 25 mL tube and grow to a | ||
+ | final OD600 of 0.2-0.4.</li> | ||
+ | <li>Then evacuate the headspace and flush with argon.</li> | ||
+ | </ol> | ||
+ | <br> | ||
+ | <a name="16"></a><span style="font-weight: bold;">Preparation for Gel | ||
+ | Electrophoresis </span><br> | ||
+ | <ol> | ||
+ | <li>Select an appropriately sized plate for the number of samples you | ||
+ | would like to run</li> | ||
+ | <li>Place the two ends into the plate to prevent the liquid gel from | ||
+ | running</li> | ||
+ | <li>Prepare the gel as follows: for a 1% gel, pour 100 mL of 1X TAE | ||
+ | and 1g of agarose into a 250 mL flask</li> | ||
+ | <li>Microwave until it begins to bubble.</li> | ||
+ | <li>Remove with a glove or hot hand and swirl until it becomes sort | ||
+ | of cloudy again</li> | ||
+ | <li>Repeat steps 4 and 5 until the liquid is clear upon removal from | ||
+ | microwave</li> | ||
+ | <li>Then add 1.5 μL of ethidium bromide for every 50 mL of gel | ||
+ | solution--may want to allow the flask to cool to the touch before | ||
+ | adding. Swirl to mix. </li> | ||
+ | <li>Set the comb for the appropriate number of lanes into the | ||
+ | plate--be sure that the comb does not touch the bottom of the plate</li> | ||
+ | <li>Pour the gel</li> | ||
+ | <li>Allow to set for about 30 minutes.</li> | ||
+ | </ol> | ||
+ | <br> | ||
+ | <a name="17"></a><span style="font-weight: bold;">CPEC (Circular | ||
+ | Polymerase Extension | ||
+ | Cloning) Protocol – Andrew Ng</span><br> | ||
+ | <ol> | ||
+ | <li>Design primers that include overhangs for fragments that you want | ||
+ | to assemble by hand or using a program such as Snapgene (these primers | ||
+ | can also be used for Gibson assembly)</li> | ||
+ | <li>Using Phusion polymerase, PCR each insert as well as your plasmid | ||
+ | backbone and verify on a gel.</li> | ||
+ | <ol> | ||
+ | <li>IMPORTANT- it is highly recommended that you optimize your PCR | ||
+ | protocol (either using temperature gradient or DMSO) so that only one | ||
+ | band can be seen in your PCR</li> | ||
+ | </ol> | ||
+ | <li>Digest PCR product using DpnI (digests methylated DNA)</li> | ||
+ | <ol> | ||
+ | <li>If PCR was performed using Phusion polymerase from NEB, you can</li> | ||
+ | </ol> | ||
+ | <li>Add the DpnI enzyme from NEB directly. For this, I add 1 ul of | ||
+ | enzyme to 50 ul of PCR product and incubate for 3 hours or more 4. PCR | ||
+ | purify your inserts,</li> | ||
+ | <ol> | ||
+ | <li>NOTE: if you were unable to get a single band in your PCR, you | ||
+ | might consider gel purification if you can get enough yield</li> | ||
+ | </ol> | ||
+ | <li>Using a 2:1 molar ratio of insert to vector (if you did not PCR | ||
+ | purify, I would just eyeball concentrations based on length of parts | ||
+ | and darkness of band in gel), make your CPEC reaction as follows:</li> | ||
+ | <ol> | ||
+ | <li>4 ul HF Buffer</li> | ||
+ | <li>0.4 ul dNTPs</li> | ||
+ | <li>.1 ul Phusion Polymerase</li> | ||
+ | <li>Insert</li> | ||
+ | <li>Vector</li> | ||
+ | <li>Fill to 20 ul with dH20</li> | ||
+ | </ol> | ||
+ | <li>(Note for this step: The more DNA you have available to use here | ||
+ | the better. I would recommend using a maximum of between 8-10 ul of DNA | ||
+ | in total though due to the salt content)</li> | ||
+ | <li>Use the following thermal cycler protocol (or just navigate to | ||
+ | CPEC in the folder labeled Andrew in the BioRad C1000)</li> | ||
+ | <ol> | ||
+ | <li>98.0C for 30s</li> | ||
+ | <li>98.0C for 10s</li> | ||
+ | <li>Ramp to 55.0C at a rate of 0.1C/s</li> | ||
+ | <li>55.0C for 30s</li> | ||
+ | <li>72.0 C for 30s/kb of construct</li> | ||
+ | <li>Repeat 1-5 for 10-30 cycles (depending on the “complexity” of | ||
+ | your assembly)</li> | ||
+ | <li>g. 72.0 C for 10 min</li> | ||
+ | </ol> | ||
+ | <li>Transform cells with 2-5ul of CPEC mix</li> | ||
+ | </ol> | ||
+ | </body> | ||
</td> | </td> |
Latest revision as of 21:33, 17 October 2014