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− | <div align="center" style="margin: 10px 0px 10px 0px; border:10px groove black;" onClick="ShowHide(cellprep)"><h3> | + | <div align="center" style="margin: 10px 0px 10px 0px; border:10px groove black;" onClick="ShowHide(cellprep)"><h3 style="text-align:center;"> Competency and transformation of <i>E. coli</i></h3></div> |
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− | <div align="center" style="margin: 10px 0px 10px; border:10px groove black;" onClick="ShowHide(compet)"><h3> | + | <div align="center" style="margin: 10px 0px 10px; border:10px groove black;" onClick="ShowHide(compet)"><h3 style="text-align:center;">Competency and transformation of <i>B. subtilis.</i></h3></div> |
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<h3>Preparation of <i>B. subtilis</i> competent cells</h3> | <h3>Preparation of <i>B. subtilis</i> competent cells</h3> | ||
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<p><br>Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath<br>Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently<br>In a sterile test tube add competent cell (0.2~0.5 ml) to the DNA solution (<0.1 ml) and incubate in a roller drum at 37.<br>Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.</p> | <p><br>Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath<br>Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently<br>In a sterile test tube add competent cell (0.2~0.5 ml) to the DNA solution (<0.1 ml) and incubate in a roller drum at 37.<br>Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media.</p> | ||
− | <div class="image" id="TransfoPic1" | + | <div class="image" id="TransfoPic1"></div> |
</div> | </div> | ||
− | <div align="center" style="margin: 10px 0px 10px; border:10px groove black;" onClick="ShowHide(BSub)"> | + | <div align="center" style="margin: 10px 0px 10px; border:10px groove black;" onClick="ShowHide(BSub)"><h3 style="text-align:center;">Vector modifications</h3></div> |
− | <h3>Vector modifications</h3></div> | + | |
<div id="BSub" style="display: none;"> | <div id="BSub" style="display: none;"> | ||
<h3>Digestion :</h3> | <h3>Digestion :</h3> | ||
<p><br>Select restriction enzymes to digest your DNA. Determine an appropriate reaction buffer by reading the instructions for your enzyme.<br>In a 1.5mL tube combine the following:</p> | <p><br>Select restriction enzymes to digest your DNA. Determine an appropriate reaction buffer by reading the instructions for your enzyme.<br>In a 1.5mL tube combine the following:</p> | ||
<ol> | <ol> | ||
− | <li>➟ DNA </li> | + | <li> ➟ DNA </li> |
− | <li>➟ Restriction Enzyme(s)</li> | + | <li> ➟ Restriction Enzyme(s)</li> |
− | <li>➟ Buffer </li> | + | <li> ➟ Buffer </li> |
− | <li>➟ dH2O up to total volume</li> | + | <li> ➟ dH2O up to total volume</li> |
</ol> | </ol> | ||
<p><br>Mix gently by pipetting.<br>Incubate tube at appropriate temperature (usually 37°C) for 1 hour.<br>Always follow the manufacturer’s instructions.<br>To visualize the results of your digest, conduct gel electrophoresis</p> | <p><br>Mix gently by pipetting.<br>Incubate tube at appropriate temperature (usually 37°C) for 1 hour.<br>Always follow the manufacturer’s instructions.<br>To visualize the results of your digest, conduct gel electrophoresis</p> | ||
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<p><br>Combine the following in a PCR or Eppendorf tube:</p> | <p><br>Combine the following in a PCR or Eppendorf tube:</p> | ||
<ol> | <ol> | ||
− | <li>➟ Vector DNA</li> | + | <li> ➟ Vector DNA</li> |
− | <li>➟ Insert DNA</li> | + | <li> ➟ Insert DNA</li> |
− | <li>➟ Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)</li> | + | <li> ➟ Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)</li> |
− | <li>➟ 0.5-1μL T4 DNA Ligase</li> | + | <li> ➟ 0.5-1μL T4 DNA Ligase</li> |
− | <li>➟ H20 to a total of 10μL</li></ol> | + | <li> ➟ H20 to a total of 10μL</li></ol> |
<p><br>To dertermine the amount of DNA needed, we used the NEB calculator<br>Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).</p> | <p><br>To dertermine the amount of DNA needed, we used the NEB calculator<br>Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).</p> | ||
</div> | </div> | ||
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− | + | <div align="center" style="margin: 10px 0px 10px; border:10px groove black;" onClick="ShowHide(other)"><h3 style="text-align:center;">Other</h3></div> | |
− | <div align="center" style="margin: 10px 0px 10px; border:10px groove black;" onClick="ShowHide(other)"><h3>Other</h3></div> | + | |
<div id="other" style="display: none;"> | <div id="other" style="display: none;"> | ||
− | <p> | + | <p><b><u>Gel Extraction:</u></b><br> |
− | < | + | We followed the protocol provided in the kit, <a href="https://www.qiagen.com/fr/resources/download.aspx?id=f4ba2d24-8218-452c-ad6f-1b6f43194425&lang=en">you can dowload it here</a><br> |
− | + | We resuspended DNA in 50 uL water.</p> | |
− | < | + | |
− | < | + | |
− | < | + | |
− | + | ||
− | < | + | |
− | </ | + | |
+ | <p><b><u>Plasmid Extraction:</u></b><br> | ||
+ | We followed the quick protocol provided in the kit, <a href="https://www.neb.com/protocols/2015/12/08/quick-protocol-for-monarch-plasmid-miniprep-kit-t1010">you can see it here</a><br> | ||
+ | We resuspended DNA in 30 - 50 uL of DNase free water.</p> | ||
+ | |||
+ | <p><b><u>PCR Purification:</u></b><br> | ||
+ | We followed the quick protocol provided in the kit, <a href="https://www.qiagen.com/fr/resources/download.aspx?id=390a728a-e6fc-43f7-bf59-b12091cc4380&lang=en">you can download it here</a><br> | ||
+ | We resuspended our purified PCR products in 50 uL water.</p> | ||
+ | |||
+ | <p><b><u>PCR:</u></b> | ||
<br> | <br> | ||
+ | We followed the protocol provided by the manufacturer, <a href="https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530">you can see it here</a><br> | ||
+ | We use different cycles programs depending on what we wanted. You can see our <a href="https://2016.igem.org/Team:UPMC-Paris/Notebook">Notebook</a> for more informations.</p> | ||
+ | |||
+ | <p><b><u>Dephosphorylation of 5' -ends:</u></b> | ||
+ | <br> | ||
+ | Using Alkaline Phosphatase we use the following protocol:<br> | ||
+ | <p>1) Make a mix of 1 pmol of DNA (about 1 ug of 3kb plasmid), 2 uL of Phosphatase Buffer, 5 units of Phosphatase and water to reach 20 uL</p> | ||
+ | <p>2) Incubats 30 minutes, 37°C</p> | ||
+ | <p>Heat inactivation at 80°C for 20 minutes</p> | ||
+ | <br> | ||
+ | <p><b><u>Ligation:</u></b> | ||
+ | <br> | ||
+ | Using T4 DNA Ligase we perform this protocol:<br> | ||
+ | <u>Reaction setup</u></p> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Component</th> | ||
+ | <th>Voplume (μl)</th> | ||
+ | <th>Final Concentration</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10X T4 DNA Ligase Buffer</td> | ||
+ | <td>2 μl</td> | ||
+ | <td> 1X </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Vector 10 1x</td> | ||
+ | <td>X μl</td> | ||
+ | <td>1-10 ng/µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Insert</td> | ||
+ | <td>X μl</td> | ||
+ | <td>1-10 ng/µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase (120 U/μl)</td> | ||
+ | <td>1 μl</td> | ||
+ | <td>6 U/µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sterile water</td> | ||
+ | <td>X μl</td> | ||
+ | <td>N/A</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total Volume</td> | ||
+ | <td>20 μl</td> | ||
+ | </tr></table> | ||
+ | <p> | ||
+ | Total reaction volume can be adjusted as needed.<br></p> | ||
+ | <ol> | ||
+ | <li> ➟ Add all of above components to a clean reaction vessel, mix well by pipetting.</li> | ||
+ | <li> ➟ Incubate at 25 °C for 30 minutes.</li> | ||
+ | <li> ➟ Immediately purify DNA using PCR clean-up column and elute in ~50 µL.</li> | ||
+ | <li> ➟ OR - Immediately dilute (at least 1:10, but enough such that 0.1-10 ng of ligation product will be transformed in TE or water </li> | ||
+ | <li> ➟ Transform 0.1-10 ng of ligation product into chemically or electrocompetent cell line that is compatible with vector</li> | ||
+ | </ol><br> | ||
</div> | </div> | ||
+ | </div> | ||
+ | <br> | ||
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 00:06, 20 October 2016