Difference between revisions of "Team:Pasteur Paris/Experiments"
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<img src="https://static.igem.org/mediawiki/2015/c/c5/Experiments_%26_Protocols_pasteur2015.jpg" style="width: 100%;"/> | <img src="https://static.igem.org/mediawiki/2015/c/c5/Experiments_%26_Protocols_pasteur2015.jpg" style="width: 100%;"/> | ||
− | + | <br/><br/> | |
+ | <div class="carregris"> | ||
<h3> Polymerase Chain Reaction </h3> | <h3> Polymerase Chain Reaction </h3> | ||
− | <h4> 1) PCR Amplification using | + | <div class="row"> |
+ | <div class="col-lg-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/e/ed/IGEM-Pasteur_PCR-PreMix.jpg" style="width:250px;height:250px"/> | ||
+ | </div> | ||
+ | <div class="col-lg-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/4/49/Igem-Pasteur_PCR.jpg" style="width:450px;height:300px" /> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <h4> 1) PCR Amplification using TaKaRa Ex Taq DNA polymerase</h4> | ||
<p> | <p> | ||
<ul> | <ul> | ||
<li>In a 0.2ml tube, set up the following reaction: </li> | <li>In a 0.2ml tube, set up the following reaction: </li> | ||
+ | <center> | ||
<table border="1" cellspacing="0" cellpadding="0" width="599"> | <table border="1" cellspacing="0" cellpadding="0" width="599"> | ||
<tr> | <tr> | ||
Line 24: | Line 35: | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Ex taq Buffer (10X)</p></td> | <td width="87" valign="top"><p align="center">Ex taq Buffer (10X)</p></td> | ||
− | <td width="104" valign="top"><p align="center"> | + | <td width="104" valign="top"><p align="center">5 µl</p></td> |
− | <td width="85" valign="top"><p align="center"> | + | <td width="85" valign="top"><p align="center">5 µl</p></td> |
− | <td width="99" valign="top"><p align="center"> | + | <td width="99" valign="top"><p align="center">5 µl</p></td> |
− | <td width="111" valign="top"><p align="center"> | + | <td width="111" valign="top"><p align="center">5 µl</p></td> |
− | <td width="113" valign="top"><p align="center"> | + | <td width="113" valign="top"><p align="center">5 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">dNTP mix</p></td> | <td width="87" valign="top"><p align="center">dNTP mix</p></td> | ||
− | <td width="104" valign="top"><p align="center"> | + | <td width="104" valign="top"><p align="center">4 µl</p></td> |
− | <td width="85" valign="top"><p align="center"> | + | <td width="85" valign="top"><p align="center">4 µl</p></td> |
− | <td width="99" valign="top"><p align="center"> | + | <td width="99" valign="top"><p align="center">4 µl</p></td> |
− | <td width="111" valign="top"><p align="center"> | + | <td width="111" valign="top"><p align="center">4 µl</p></td> |
− | <td width="113" valign="top"><p align="center"> | + | <td width="113" valign="top"><p align="center">4 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Template DNA</p></td> | <td width="87" valign="top"><p align="center">Template DNA</p></td> | ||
− | <td width="104" valign="top"><p align="center">1- | + | <td width="104" valign="top"><p align="center">1-10 ng</p></td> |
− | <td width="85" valign="top"><p align="center">1- | + | <td width="85" valign="top"><p align="center">1-10 ng</p></td> |
− | <td width="99" valign="top"><p align="center">1- | + | <td width="99" valign="top"><p align="center">1-10 ng</p></td> |
− | <td width="111" valign="top"><p align="center">1- | + | <td width="111" valign="top"><p align="center">1-10 ng</p></td> |
<td width="113" valign="top"><p> </p></td> | <td width="113" valign="top"><p> </p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Forward Primer</p></td> | <td width="87" valign="top"><p align="center">Forward Primer</p></td> | ||
− | <td width="104" valign="top"><p align="center">final concentration: 0. | + | <td width="104" valign="top"><p align="center">final concentration: 0.2 µM</p></td> |
<td width="85" valign="top"><p> </p></td> | <td width="85" valign="top"><p> </p></td> | ||
− | <td width="99" valign="top"><p align="center">final concentration: 0. | + | <td width="99" valign="top"><p align="center">final concentration: 0.2 µM</p></td> |
<td width="111" valign="top"><p> </p></td> | <td width="111" valign="top"><p> </p></td> | ||
− | <td width="113" valign="top"><p align="center">final concentration: 0. | + | <td width="113" valign="top"><p align="center">final concentration: 0.2 µM</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Reverse Primer</p></td> | <td width="87" valign="top"><p align="center">Reverse Primer</p></td> | ||
− | <td width="104" valign="top"><p align="center">final concentration: 0. | + | <td width="104" valign="top"><p align="center">final concentration: 0.2 µM</p></td> |
<td width="85" valign="top"><p> </p></td> | <td width="85" valign="top"><p> </p></td> | ||
<td width="99" valign="top"><p> </p></td> | <td width="99" valign="top"><p> </p></td> | ||
− | <td width="111" valign="top"><p align="center">final concentration: 0. | + | <td width="111" valign="top"><p align="center">final concentration: 0.2 µM</p></td> |
− | <td width="113" valign="top"><p align="center">final concentration: 0. | + | <td width="113" valign="top"><p align="center">final concentration: 0.2 µM</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Nuclease free water</p></td> | <td width="87" valign="top"><p align="center">Nuclease free water</p></td> | ||
− | <td width="104" valign="top"><p align="center">to | + | <td width="104" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="85" valign="top"><p align="center">to | + | <td width="85" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="99" valign="top"><p align="center">to | + | <td width="99" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="111" valign="top"><p align="center">to | + | <td width="111" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="113" valign="top"><p align="center">to | + | <td width="113" valign="top"><p align="center">to 50 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Ex Taq DNA Polymerase</p></td> | <td width="87" valign="top"><p align="center">Ex Taq DNA Polymerase</p></td> | ||
− | <td width="104" valign="top"><p align="center">0. | + | <td width="104" valign="top"><p align="center">0.25 µl</p></td> |
− | <td width="85" valign="top"><p align="center">0. | + | <td width="85" valign="top"><p align="center">0.25 µl</p></td> |
− | <td width="99" valign="top"><p align="center">0. | + | <td width="99" valign="top"><p align="center">0.25 µl</p></td> |
− | <td width="111" valign="top"><p align="center">0. | + | <td width="111" valign="top"><p align="center">0.25 µl</p></td> |
− | <td width="113" valign="top"><p align="center">0. | + | <td width="113" valign="top"><p align="center">0.25 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Total</p></td> | <td width="87" valign="top"><p align="center">Total</p></td> | ||
− | <td width="104" valign="top"><p align="center"> | + | <td width="104" valign="top"><p align="center">50 µl</p></td> |
− | <td width="85" valign="top"><p align="center"> | + | <td width="85" valign="top"><p align="center">50 µl</p></td> |
− | <td width="99" valign="top"><p align="center"> | + | <td width="99" valign="top"><p align="center">50 µl</p></td> |
− | <td width="111" valign="top"><p align="center"> | + | <td width="111" valign="top"><p align="center">50 µl</p></td> |
− | <td width="113" valign="top"><p align="center"> | + | <td width="113" valign="top"><p align="center">50 µl</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
<li> Set up the following cycles in a PCR machine | <li> Set up the following cycles in a PCR machine | ||
<ul> | <ul> | ||
− | <li>Initial denaturation : 94°C for 30 sec</li> | + | <li>Initial denaturation: 94°C for 30 sec</li> |
<li>30 cycles : </li> | <li>30 cycles : </li> | ||
− | <ul><li> 94°C for | + | <ul><li> 94°C for 30 sec</li> |
<li>55°C - 65°C for 1 min depending on your annealing temperature</li> | <li>55°C - 65°C for 1 min depending on your annealing temperature</li> | ||
<li>72°C 0.5-1 min per kb</li></ul> | <li>72°C 0.5-1 min per kb</li></ul> | ||
− | <li> Final extension: 72°C for | + | <li> Final extension: 72°C for 5 min.</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
Line 106: | Line 120: | ||
<li>In a 0.2ml tube, set up the following reaction: </li> | <li>In a 0.2ml tube, set up the following reaction: </li> | ||
+ | <center> | ||
<table border="1" cellspacing="0" cellpadding="0" width="599"> | <table border="1" cellspacing="0" cellpadding="0" width="599"> | ||
<tr> | <tr> | ||
Line 117: | Line 132: | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Phusion HF Buffer (5X)</p></td> | <td width="87" valign="top"><p align="center">Phusion HF Buffer (5X)</p></td> | ||
− | <td width="104" valign="top"><p align="center"> | + | <td width="104" valign="top"><p align="center">10 µl</p></td> |
− | <td width="85" valign="top"><p align="center"> | + | <td width="85" valign="top"><p align="center">10 µl</p></td> |
− | <td width="99" valign="top"><p align="center"> | + | <td width="99" valign="top"><p align="center">10 µl</p></td> |
− | <td width="111" valign="top"><p align="center"> | + | <td width="111" valign="top"><p align="center">10 µl</p></td> |
− | <td width="113" valign="top"><p align="center"> | + | <td width="113" valign="top"><p align="center">10 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">dNTPs (10mM)</p></td> | <td width="87" valign="top"><p align="center">dNTPs (10mM)</p></td> | ||
− | <td width="104" valign="top"><p align="center"> | + | <td width="104" valign="top"><p align="center">1 µl</p></td> |
− | <td width="85" valign="top"><p align="center"> | + | <td width="85" valign="top"><p align="center">1 µl</p></td> |
− | <td width="99" valign="top"><p align="center"> | + | <td width="99" valign="top"><p align="center">1 µl</p></td> |
<td width="111" valign="top"><p align="center">1µl</p></td> | <td width="111" valign="top"><p align="center">1µl</p></td> | ||
− | <td width="113" valign="top"><p align="center"> | + | <td width="113" valign="top"><p align="center">1 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Template DNA</p></td> | <td width="87" valign="top"><p align="center">Template DNA</p></td> | ||
− | <td width="104" valign="top"><p align="center">< | + | <td width="104" valign="top"><p align="center"><250 ng</p></td> |
− | <td width="85" valign="top"><p align="center">< | + | <td width="85" valign="top"><p align="center"><250 ng</p></td> |
− | <td width="99" valign="top"><p align="center">< | + | <td width="99" valign="top"><p align="center"><250 ng</p></td> |
− | <td width="111" valign="top"><p align="center">< | + | <td width="111" valign="top"><p align="center"><250 ng</p></td> |
<td width="113" valign="top"><p> </p></td> | <td width="113" valign="top"><p> </p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">10µM forward Primer</p></td> | <td width="87" valign="top"><p align="center">10µM forward Primer</p></td> | ||
− | <td width="104" valign="top"><p align="center">2. | + | <td width="104" valign="top"><p align="center">2.5 µl</p></td> |
<td width="85" valign="top"><p> </p></td> | <td width="85" valign="top"><p> </p></td> | ||
− | <td width="99" valign="top"><p align="center">2. | + | <td width="99" valign="top"><p align="center">2.5 µl</p></td> |
<td width="111" valign="top"><p> </p></td> | <td width="111" valign="top"><p> </p></td> | ||
− | <td width="113" valign="top"><p align="center">2. | + | <td width="113" valign="top"><p align="center">2.5 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">10µM reverse primer</p></td> | <td width="87" valign="top"><p align="center">10µM reverse primer</p></td> | ||
− | <td width="104" valign="top"><p align="center">2. | + | <td width="104" valign="top"><p align="center">2.5 µl</p></td> |
<td width="85" valign="top"><p> </p></td> | <td width="85" valign="top"><p> </p></td> | ||
<td width="99" valign="top"><p> </p></td> | <td width="99" valign="top"><p> </p></td> | ||
− | <td width="111" valign="top"><p align="center">2. | + | <td width="111" valign="top"><p align="center">2.5 µl</p></td> |
− | <td width="113" valign="top"><p align="center">2. | + | <td width="113" valign="top"><p align="center">2.5 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Nuclease free water</p></td> | <td width="87" valign="top"><p align="center">Nuclease free water</p></td> | ||
− | <td width="104" valign="top"><p align="center">to | + | <td width="104" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="85" valign="top"><p align="center">to | + | <td width="85" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="99" valign="top"><p align="center">to | + | <td width="99" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="111" valign="top"><p align="center">to | + | <td width="111" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="113" valign="top"><p align="center">to | + | <td width="113" valign="top"><p align="center">to 50 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Phusion DNA Polymerase</p></td> | <td width="87" valign="top"><p align="center">Phusion DNA Polymerase</p></td> | ||
− | <td width="104" valign="top"><p align="center">0. | + | <td width="104" valign="top"><p align="center">0.5 µl</p></td> |
− | <td width="85" valign="top"><p align="center">0. | + | <td width="85" valign="top"><p align="center">0.5 µl</p></td> |
− | <td width="99" valign="top"><p align="center">0. | + | <td width="99" valign="top"><p align="center">0.5 µl</p></td> |
− | <td width="111" valign="top"><p align="center">0. | + | <td width="111" valign="top"><p align="center">0.5 µl</p></td> |
− | <td width="113" valign="top"><p align="center">0. | + | <td width="113" valign="top"><p align="center">0.5 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Total</p></td> | <td width="87" valign="top"><p align="center">Total</p></td> | ||
− | <td width="104" valign="top"><p align="center"> | + | <td width="104" valign="top"><p align="center">50 µl</p></td> |
− | <td width="85" valign="top"><p align="center"> | + | <td width="85" valign="top"><p align="center">50 µl</p></td> |
− | <td width="99" valign="top"><p align="center"> | + | <td width="99" valign="top"><p align="center">50 µl</p></td> |
− | <td width="111" valign="top"><p align="center"> | + | <td width="111" valign="top"><p align="center">50 µl</p></td> |
− | <td width="113" valign="top"><p align="center"> | + | <td width="113" valign="top"><p align="center">50 µl</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
<li> Set up the following cycles in a PCR machine | <li> Set up the following cycles in a PCR machine | ||
Line 186: | Line 204: | ||
<li>30 cycles : </li> | <li>30 cycles : </li> | ||
<ul><li> 94°C for 5-10s</li> | <ul><li> 94°C for 5-10s</li> | ||
− | <li>45°C - 72°C for 10 to | + | <li>45°C - 72°C for 10 to 30 sec depending on your annealing temperature</li> |
− | <li>72°C for 15- | + | <li>72°C for 15-30 sec per kb</li></ul> |
− | <li> Final extension: 72°C for | + | <li> Final extension: 72°C for 5 min.</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
Line 199: | Line 217: | ||
<li>In a 0.2ml tube, set up the following reaction: </li> | <li>In a 0.2ml tube, set up the following reaction: </li> | ||
+ | <center> | ||
<table border="1" cellspacing="0" cellpadding="0" width="599"> | <table border="1" cellspacing="0" cellpadding="0" width="599"> | ||
<tr> | <tr> | ||
Line 210: | Line 229: | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Q5 HIgh Fidelity Master Mix (2X)</p></td> | <td width="87" valign="top"><p align="center">Q5 HIgh Fidelity Master Mix (2X)</p></td> | ||
− | <td width="104" valign="top"><p align="center"> | + | <td width="104" valign="top"><p align="center">25 µl</p></td> |
− | <td width="85" valign="top"><p align="center"> | + | <td width="85" valign="top"><p align="center">25 µl</p></td> |
− | <td width="99" valign="top"><p align="center"> | + | <td width="99" valign="top"><p align="center">25 µl</p></td> |
− | <td width="111" valign="top"><p align="center"> | + | <td width="111" valign="top"><p align="center">25 µl</p></td> |
− | <td width="113" valign="top"><p align="center"> | + | <td width="113" valign="top"><p align="center">25 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Template DNA</p></td> | <td width="87" valign="top"><p align="center">Template DNA</p></td> | ||
− | <td width="104" valign="top"><p align="center">< | + | <td width="104" valign="top"><p align="center"><1 ng</p></td> |
− | <td width="85" valign="top"><p align="center">< | + | <td width="85" valign="top"><p align="center"><1 ng</p></td> |
− | <td width="99" valign="top"><p align="center">< | + | <td width="99" valign="top"><p align="center"><1 ng</p></td> |
− | <td width="111" valign="top"><p align="center">< | + | <td width="111" valign="top"><p align="center"><1 ng</p></td> |
− | <td width="113" valign="top"><p align="center">< | + | <td width="113" valign="top"><p align="center"><1 ng</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width="87" valign="top"><p align="center"> | + | <td width="87" valign="top"><p align="center">10 µM forward Primer</p></td> |
− | <td width="104" valign="top"><p align="center">2. | + | <td width="104" valign="top"><p align="center">2.5 µl</p></td> |
<td width="85" valign="top"><p> </p></td> | <td width="85" valign="top"><p> </p></td> | ||
− | <td width="99" valign="top"><p align="center">2. | + | <td width="99" valign="top"><p align="center">2.5 µl</p></td> |
<td width="111" valign="top"><p> </p></td> | <td width="111" valign="top"><p> </p></td> | ||
− | <td width="113" valign="top"><p align="center">2. | + | <td width="113" valign="top"><p align="center">2.5 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width="87" valign="top"><p align="center"> | + | <td width="87" valign="top"><p align="center">10 µM reverse primer</p></td> |
− | <td width="104" valign="top"><p align="center">2. | + | <td width="104" valign="top"><p align="center">2.5 µl</p></td> |
<td width="85" valign="top"><p> </p></td> | <td width="85" valign="top"><p> </p></td> | ||
<td width="99" valign="top"><p> </p></td> | <td width="99" valign="top"><p> </p></td> | ||
− | <td width="111" valign="top"><p align="center">2. | + | <td width="111" valign="top"><p align="center">2.5 µl</p></td> |
− | <td width="113" valign="top"><p align="center">2. | + | <td width="113" valign="top"><p align="center">2.5 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Nuclease free water</p></td> | <td width="87" valign="top"><p align="center">Nuclease free water</p></td> | ||
− | <td width="104" valign="top"><p align="center">to | + | <td width="104" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="85" valign="top"><p align="center">to | + | <td width="85" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="99" valign="top"><p align="center">to | + | <td width="99" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="111" valign="top"><p align="center">to | + | <td width="111" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="113" valign="top"><p align="center">to | + | <td width="113" valign="top"><p align="center">to 50 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="87" valign="top"><p align="center">Total</p></td> | <td width="87" valign="top"><p align="center">Total</p></td> | ||
− | <td width="104" valign="top"><p align="center"> | + | <td width="104" valign="top"><p align="center">50 µl</p></td> |
− | <td width="85" valign="top"><p align="center"> | + | <td width="85" valign="top"><p align="center">50 µl</p></td> |
− | <td width="99" valign="top"><p align="center"> | + | <td width="99" valign="top"><p align="center">50 µl</p></td> |
− | <td width="111" valign="top"><p align="center"> | + | <td width="111" valign="top"><p align="center">50 µl</p></td> |
− | <td width="113" valign="top"><p align="center"> | + | <td width="113" valign="top"><p align="center">50 µl</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
+ | </center> | ||
+ | <br> | ||
<li> Set up the following cycles in a PCR machine | <li> Set up the following cycles in a PCR machine | ||
Line 264: | Line 285: | ||
<li>30 cycles : </li> | <li>30 cycles : </li> | ||
<ul><li> 94°C for 5-10s</li> | <ul><li> 94°C for 5-10s</li> | ||
− | <li> 45°C - 72°C for 10 to | + | <li> 45°C - 72°C for 10 to 30 sec depending on your annealing temperature</li> |
− | <li>72°C for 20- | + | <li>72°C for 20-30 sec per kb</li></ul> |
− | <li> Final extension: 72°C for | + | <li> Final extension: 72°C for 2 min.</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
+ | <br/><div class="carregris"> | ||
<h3> Enzymatic Digestion </h3> | <h3> Enzymatic Digestion </h3> | ||
+ | |||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/8/87/Igem-Pasteur_DigestionMix.jpg", height=400px /> | ||
+ | </center> | ||
+ | |||
<h4> 1) Single restriction enzyme digestion</h4> | <h4> 1) Single restriction enzyme digestion</h4> | ||
<p> | <p> | ||
Line 277: | Line 305: | ||
<li>In a MicroCentrifuge tube, set up the following reaction on ice: </li> | <li>In a MicroCentrifuge tube, set up the following reaction on ice: </li> | ||
+ | <center> | ||
<table border="1" cellspacing="0" cellpadding="0" width="344"> | <table border="1" cellspacing="0" cellpadding="0" width="344"> | ||
<tr> | <tr> | ||
Line 285: | Line 314: | ||
<tr> | <tr> | ||
<td width="174" valign="top"><p align="center">Buffer (10X)</p></td> | <td width="174" valign="top"><p align="center">Buffer (10X)</p></td> | ||
− | <td width="83" valign="top"><p align="center"> | + | <td width="83" valign="top"><p align="center">5 µl</p></td> |
− | <td width="87" valign="top"><p align="center"> | + | <td width="87" valign="top"><p align="center">5 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="174" valign="top"><p align="center">DNA</p></td> | <td width="174" valign="top"><p align="center">DNA</p></td> | ||
− | <td width="83" valign="top"><p align="center"> | + | <td width="83" valign="top"><p align="center">1 µg</p></td> |
− | <td width="87" valign="top"><p align="center"> | + | <td width="87" valign="top"><p align="center">1 µg</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="174" valign="top"><p align="center">Restriction Enzyme</p></td> | <td width="174" valign="top"><p align="center">Restriction Enzyme</p></td> | ||
− | <td width="83" valign="top"><p align="center"> | + | <td width="83" valign="top"><p align="center">1 µl</p></td> |
<td width="87" valign="top"><p> </p></td> | <td width="87" valign="top"><p> </p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width="174" valign="top"><p align="center">DNAse, RNAse | + | <td width="174" valign="top"><p align="center">DNAse, RNAse free water</p></td> |
− | <td width="83" valign="top"><p align="center">to | + | <td width="83" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="87" valign="top"><p align="center">to | + | <td width="87" valign="top"><p align="center">to 50 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="174" valign="top"><p align="center">Total</p></td> | <td width="174" valign="top"><p align="center">Total</p></td> | ||
− | <td width="83" valign="top"><p align="center"> | + | <td width="83" valign="top"><p align="center">50 µl</p></td> |
− | <td width="87" valign="top"><p align="center"> | + | <td width="87" valign="top"><p align="center">50 µl</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
<li>Pipette up and down to homogenize the solution.</li> | <li>Pipette up and down to homogenize the solution.</li> | ||
− | <li>Quick spin in a MicroCentrifuge ( | + | <li>Quick spin in a MicroCentrifuge (5 sec).</li> |
<li>Incubation at 37°C for 1h.</li> | <li>Incubation at 37°C for 1h.</li> | ||
− | <li>Heat inactivation for | + | <li>Heat inactivation for 20 min at 80°C.</li> |
<b>Optional</b> | <b>Optional</b> | ||
− | <li>Add the phosphatase: Add 1 unit of Shrimp | + | <li>Add the phosphatase: Add 1 unit of Shrimp alkaline phosphatase for each pmol of phosphate end.</li> |
− | <li>Incubate for | + | <li>Incubate for 30 min at 37°C. </li> |
<li>Inactivate the phosphatase at 65°C for 15 min. </li> | <li>Inactivate the phosphatase at 65°C for 15 min. </li> | ||
</ul> | </ul> | ||
Line 325: | Line 357: | ||
<ul> | <ul> | ||
<li>In a MicroCentrifuge tube, set up the following reaction on ice: </li> | <li>In a MicroCentrifuge tube, set up the following reaction on ice: </li> | ||
+ | <center> | ||
<table border="1" cellspacing="0" cellpadding="0" width="344"> | <table border="1" cellspacing="0" cellpadding="0" width="344"> | ||
<tr> | <tr> | ||
Line 336: | Line 369: | ||
<tr> | <tr> | ||
<td width="99" valign="top"><p align="center">Buffer (10X)</p></td> | <td width="99" valign="top"><p align="center">Buffer (10X)</p></td> | ||
− | <td width="47" valign="top"><p align="center"> | + | <td width="47" valign="top"><p align="center">5 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">5 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">5 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">5 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">5 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="99" valign="top"><p align="center">DNA</p></td> | <td width="99" valign="top"><p align="center">DNA</p></td> | ||
− | <td width="47" valign="top"><p align="center"> | + | <td width="47" valign="top"><p align="center">1 µg</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">1 µg</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">1 µg</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">1 µg</p></td> |
<td width="49" valign="top"><p align="center">0</p></td> | <td width="49" valign="top"><p align="center">0</p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="99" valign="top"><p align="center">Restriction Enzyme n°1</p></td> | <td width="99" valign="top"><p align="center">Restriction Enzyme n°1</p></td> | ||
− | <td width="47" valign="top"><p align="center"> | + | <td width="47" valign="top"><p align="center">1 µl</p></td> |
<td width="49" valign="top"><p> </p></td> | <td width="49" valign="top"><p> </p></td> | ||
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">1 µl</p></td> |
<td width="49" valign="top"><p> </p></td> | <td width="49" valign="top"><p> </p></td> | ||
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">1 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="99" valign="top"><p align="center">Restriction Enzyme n°2</p></td> | <td width="99" valign="top"><p align="center">Restriction Enzyme n°2</p></td> | ||
− | <td width="47" valign="top"><p align="center"> | + | <td width="47" valign="top"><p align="center">1 µl</p></td> |
<td width="49" valign="top"><p> </p></td> | <td width="49" valign="top"><p> </p></td> | ||
<td width="49" valign="top"><p> </p></td> | <td width="49" valign="top"><p> </p></td> | ||
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">1 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">1 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="99" valign="top"><p align="center">Nuclease free water</p></td> | <td width="99" valign="top"><p align="center">Nuclease free water</p></td> | ||
− | <td width="47" valign="top"><p align="center">to | + | <td width="47" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="49" valign="top"><p align="center">to | + | <td width="49" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="49" valign="top"><p align="center">to | + | <td width="49" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="49" valign="top"><p align="center">to | + | <td width="49" valign="top"><p align="center">to 50 µl</p></td> |
− | <td width="49" valign="top"><p align="center">to | + | <td width="49" valign="top"><p align="center">to 50 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td width="99" valign="top"><p align="center">Total</p></td> | <td width="99" valign="top"><p align="center">Total</p></td> | ||
− | <td width="47" valign="top"><p align="center"> | + | <td width="47" valign="top"><p align="center">50 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">50 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">50 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">50 µl</p></td> |
− | <td width="49" valign="top"><p align="center"> | + | <td width="49" valign="top"><p align="center">50 µl</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
<li>Pipette up and down to homogenize the solution.</li> | <li>Pipette up and down to homogenize the solution.</li> | ||
− | <li>Quick spin in a | + | <li>Quick spin in a Micro-centrifuge (5 sec).</li> |
<li>Incubation at 37°C for 1h.</li> | <li>Incubation at 37°C for 1h.</li> | ||
− | <li>Heat inactivation for | + | <li>Heat inactivation for 20 min at 80°C.</li> |
<b>Optional</b> | <b>Optional</b> | ||
− | <li>Add the phosphatase: Add 1 unit of Shrimp | + | <li>Add the phosphatase: Add 1 unit of Shrimp alkaline phosphatase for each pmol of phosphate end.</li> |
− | <li>Incubate for | + | <li>Incubate for 30 min at 37°C. </li> |
<li>Inactivate the phosphatase at 65°C for 15 min. </li> | <li>Inactivate the phosphatase at 65°C for 15 min. </li> | ||
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
+ | <br/><div class="carregris"> | ||
+ | <h3> Agarose Gel Electrophoresis </h3> | ||
+ | |||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/a/a8/IGEM-Pasteur_illustration-Protocols.jpg" /> | ||
+ | </center> | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Prepare the different dilutions of TAE Buffer (50X, 1X, 0.5X) </li> | ||
+ | <li>Agarose gel preparation : </li> | ||
+ | <ul> | ||
+ | <li>Weigh the agarose (Invitrogen Ref 16500-500) depending on the size of your DNA.</li> | ||
+ | <li>Dissolve it in the appropriate amount of TAE Buffer (1X). </li> | ||
+ | <li>Heat the preparation in the microwave until the agarose is dissolved. </li> | ||
+ | <li>Cool the preparation by letting cool water flow against the Erlenmeyer until there is no evaporation. </li> | ||
+ | <li>Add 1 drop of EB (Eurobio Ref GEPBET02-AF) under the extraction hood. Mix gently. </li> | ||
+ | <li>Pour the solution in the casting tray and remove any bubbles. </li> | ||
+ | <li>Place the combs in the casting tray and let it rest until the gel is solid. </li> | ||
+ | <li>Carefully remove the combs from the gel </li> | ||
+ | </ul> | ||
+ | <li>Place the agarose gel in the electrophoresis apparatus filled with TAE buffer (0.5X). </li> | ||
+ | <li>Gel loading: </li> | ||
+ | <ul> | ||
+ | <li>Load 2 µl of DNA ladder in the first and eventually the last well. </li> | ||
+ | <li>Load each well with the appropriate amount of DNA and loading buffer. </li> | ||
+ | </ul> | ||
+ | <li>Close the electrophoresis unit and run the gel for 1 hour at 130 V and 7 mA. </li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <br/><div class="carregris"> | ||
+ | <h3>QIAgen Gel Extraction Kit Protocol</h3> | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. </li> | ||
+ | <li>Weigh the gel slice in a colorless tube</li> | ||
+ | <li>Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 μl). For >2% agarose gels, add 6 volumes Buffer QG.</li> | ||
+ | <li>Incubate at 50°C for until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel.</li> | ||
+ | <li>Check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.</li> | ||
+ | <li>Add 1 gel volume of isopropanol to the sample and mix.</li> | ||
+ | <li>Place a QIAquick spin column in a provided 2 ml collection tube.</li> | ||
+ | <li>Apply the sample to the QIAquick column and centrifuge for 1 min.</li> | ||
+ | <li>Discard flow-through and place the QIAquick column back into the same tube. </li> | ||
+ | <li>To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 1 min</li> | ||
+ | <li>Discard flow-through and place the QIAquick column back into the same tube.</li> | ||
+ | <li>Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 17,900 x g to remove residual wash buffer.</li> | ||
+ | <li>Place QIAquick column into a clean 1.5 ml microcentrifuge tube.</li> | ||
+ | <li>To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min.</li> | ||
+ | <li>Let the column stand for 1 min,</li> | ||
+ | <li>Centrifuge for 1 min.</li> | ||
+ | <li>If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.</li> | ||
+ | |||
+ | </ul> | ||
+ | </p> | ||
+ | |||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/2/21/IGEM_Pasteur_Gel_extraction_kit.jpg" /> | ||
+ | </center> | ||
+ | </div> | ||
+ | |||
+ | <br/><div class="carregris"> | ||
<h3> Ligation</h3> | <h3> Ligation</h3> | ||
+ | |||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/e/e3/Igem-Pasteur_LigationMix.jpg" /> | ||
+ | </center> | ||
+ | |||
<p> | <p> | ||
<ul> | <ul> | ||
Line 401: | Line 505: | ||
<li> Set up the following reaction in a microcentrifuge tube on ice : </li> | <li> Set up the following reaction in a microcentrifuge tube on ice : </li> | ||
+ | <center> | ||
<table border="1" cellspacing="0" cellpadding="0" width="344"> | <table border="1" cellspacing="0" cellpadding="0" width="344"> | ||
<tr> | <tr> | ||
Line 426: | Line 531: | ||
<tr> | <tr> | ||
<td width="233" valign="top"><p align="center">Nuclease free water</p></td> | <td width="233" valign="top"><p align="center">Nuclease free water</p></td> | ||
− | <td width="111" valign="top"><p align="center">to | + | <td width="111" valign="top"><p align="center">to 20 µl</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 433: | Line 538: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
<li> Gently mix the reaction by pipetting up and down</li> | <li> Gently mix the reaction by pipetting up and down</li> | ||
− | <li>Quick spin in a MicroCentrifuge ( | + | <li>Quick spin in a MicroCentrifuge (5 sec).</li> |
<li>For cohesive ends, incubation at 16°C for 1h.</li> | <li>For cohesive ends, incubation at 16°C for 1h.</li> | ||
<li>Heat inactivation for 10min at 65°C.</li> | <li>Heat inactivation for 10min at 65°C.</li> | ||
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
+ | |||
+ | <br/><div class="carregris"> | ||
+ | <h3>Transformation in chemically competent <i>E. coli</i> DH5-α </h3> | ||
+ | |||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/8/84/Igem-Pasteur_TransformationMix.jpg", height=400px /> | ||
+ | </center> | ||
+ | |||
+ | <p> | ||
+ | <ul> | ||
+ | <li> Thaw out on ice one tube of chemically competent <i>E. coli</i> DH5-α. </li> | ||
+ | <li> Place 50µL of cells in a pre-chilled Micro-centrifuge tube. </li> | ||
+ | <li> Refreeze any unused cells. </li> | ||
+ | <li>Add 1-10ng of DNA to the cells and mix gently by tapping on the tube. Do not mix by pipetting up and down.</li> | ||
+ | <li> Incubate on ice for 30 min. </li> | ||
+ | <li> Heat shock the cells for 40 sec in a 42°C water bath. </li> | ||
+ | <li> Place the tubes on ice for 3 minutes. </li> | ||
+ | <li> Add 700 µl of pre-warmed (37°C) SOC medium (Invitrogen NO 15544-034)</li> | ||
+ | <li> Incubate at 37°C for 40 min at 200 rpm. </li> | ||
+ | <li>Spread 200 µl of transformed cells on the appropriate medium.</li> | ||
+ | <li>Incubate overnight at 37°C</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | <br/><div class="carregris"> | ||
+ | <h3> Stab Cultures</h3> | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Prepare and autoclave 0.7% LB agar (standard LB medium containing 7 g/liter agar). </li> | ||
+ | <li>Cool the LB agar to below 50°C (when you can hold it comfortably) and add the appropriate antibiotic(s). While still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify.</li> | ||
+ | <li>Using a sterile straight wire, pick a single colony from a freshly grown plate and stab it deep down into the soft agar several times. </li> | ||
+ | <li>Incubate the vial at 37°C for 8–12 h leaving the cap slightly loose. </li> | ||
+ | <li>Seal the vial tightly and store in the dark, preferably at 4°C. </li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | </div> | ||
+ | <br/><div class="carregris"> | ||
<h3> MiniPrep </h3> | <h3> MiniPrep </h3> | ||
<h4> 1) Bacterial Culture Growth</h4> | <h4> 1) Bacterial Culture Growth</h4> | ||
Line 455: | Line 602: | ||
<li>Dilute the starter culture 1/500 to 1/1000 into selective LB medium. </li> | <li>Dilute the starter culture 1/500 to 1/1000 into selective LB medium. </li> | ||
<li>Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm). </li> | <li>Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm). </li> | ||
− | <li>Centrifuge at | + | <li>Centrifuge at 6,000 x g for 15 min at 4°C. </li> |
− | <li> | + | <li>Re-suspend the bacterial pellet in 4 ml Buffer P1. </li> |
<li>Add 4 ml Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times </li> | <li>Add 4 ml Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times </li> | ||
<li>Incubate at room temperature (15–25°C) for 5 min. </li> | <li>Incubate at room temperature (15–25°C) for 5 min. </li> | ||
Line 465: | Line 612: | ||
<li>Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow. </li> | <li>Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow. </li> | ||
<li>Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow. </li> | <li>Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow. </li> | ||
− | <li>Wash the QIAGEN-tip with | + | <li>Wash the QIAGEN-tip with 2 x 10 ml Buffer QC. </li> |
− | <li>Elute DNA with | + | <li>Elute DNA with 5 ml Buffer QF. </li> |
− | <li>Collect the eluate in a 15 ml or | + | <li>Collect the eluate in a 15 ml or 50 ml tube. </li> |
<li>Add 3.5 ml (0.7 volumes) room-temperature isopropanol to the eluted DNA. </li> | <li>Add 3.5 ml (0.7 volumes) room-temperature isopropanol to the eluted DNA. </li> | ||
<li>Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C. </li> | <li>Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C. </li> | ||
<li>Carefully decant the supernatant. </li> | <li>Carefully decant the supernatant. </li> | ||
− | <li>Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of TE 8.1 Buffer | + | <li>Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of TE 8.1 Buffer (Tris 10 mM, EDTA 0.1 mM). </li> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
+ | <br/><div class="carregris"> | ||
<h3> MidiPrep </h3> | <h3> MidiPrep </h3> | ||
<h4> 1) Bacterial Culture Growth</h4> | <h4> 1) Bacterial Culture Growth</h4> | ||
Line 493: | Line 630: | ||
<li> Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic.</li> | <li> Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic.</li> | ||
<li> Incubate for 12–16 h at 37°C with vigorous shaking. </li> | <li> Incubate for 12–16 h at 37°C with vigorous shaking. </li> | ||
− | <li>Centrifugation at > | + | <li>Centrifugation at > 8,000 rpm (6,800 x g) in MicroCentrifuge for 3 min at room temperature (15–25°C).</li> |
<li> Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained. </li> | <li> Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained. </li> | ||
</ul> | </ul> | ||
Line 501: | Line 638: | ||
<p> | <p> | ||
<ul> | <ul> | ||
− | <li>Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a micro- centrifuge tube.</li> | + | <li>Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a micro-centrifuge tube.</li> |
<li>Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. </li> | <li>Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. </li> | ||
<li>Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. </li> | <li>Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. </li> | ||
<li>Centrifuge for 10 min at 13,000 rpm in a MicroCentrifuge. </li> | <li>Centrifuge for 10 min at 13,000 rpm in a MicroCentrifuge. </li> | ||
<li>Apply the supernatants to the QIAprep spin column by decanting or pipetting.</li> | <li>Apply the supernatants to the QIAprep spin column by decanting or pipetting.</li> | ||
− | <li>Centrifuge for 30–60 | + | <li>Centrifuge for 30–60 sec. Discard the flow-through. </li> |
− | <li>Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 | + | <li>Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 sec. Discard the flow-through. </li> |
<li>Add 0.75 ml Buffer PE and centrifuge for 30–60 s. </li> | <li>Add 0.75 ml Buffer PE and centrifuge for 30–60 s. </li> | ||
<li>Discard the flow-through, and centrifuge at full speed for an additional 1 min. </li> | <li>Discard the flow-through, and centrifuge at full speed for an additional 1 min. </li> | ||
<li>Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. </li> | <li>Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. </li> | ||
− | <li>Add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column. </li> | + | <li>Add 50 μl Buffer EB (Qiagen Elution buffer) (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column. </li> |
<li>Let stand for 1 min. </li> | <li>Let stand for 1 min. </li> | ||
<li>Centrifuge for 1 min. </li> | <li>Centrifuge for 1 min. </li> | ||
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
− | < | + | <br/><div class="carregris"> |
− | < | + | <h3>Preparation of Electro-competent <i>E. coli</i> BAP 1. </h3> |
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<p> All the following steps take place under sterile conditions and on ice</p> | <p> All the following steps take place under sterile conditions and on ice</p> | ||
<p> | <p> | ||
<ul> | <ul> | ||
− | <li>Inoculate 500mL of L-broth with 1/100 volume of a fresh overnight E. coli | + | <li>Inoculate 500mL of L-broth with 1/100 volume of a fresh overnight <i>E. coli</i> BAP 1 culture. </li> |
<li>Grow the cells at 37 °C at 300 rpm. </li> | <li>Grow the cells at 37 °C at 300 rpm. </li> | ||
<li>Chill cells on ice for about 20 min. </li> | <li>Chill cells on ice for about 20 min. </li> | ||
− | <li>Transfer the cells to a cold centrifuge bottle and spin at | + | <li>Transfer the cells to a cold centrifuge bottle and spin at 4,000 x g for 15 minutes at 4 °C. </li> |
<li>Carefully pour off and discard the supernatant. It is better to sacrifice a few cells than to leave supernatant behind. </li> | <li>Carefully pour off and discard the supernatant. It is better to sacrifice a few cells than to leave supernatant behind. </li> | ||
<li>Gently re-suspend the pellet in 500 ml of ice-cold glycerol (10%). </li> | <li>Gently re-suspend the pellet in 500 ml of ice-cold glycerol (10%). </li> | ||
− | <li>Centrifuge at | + | <li>Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant. </li> |
<li>Re-suspend the pellet in 250 ml of ice-cold glycerol (10%). </li> | <li>Re-suspend the pellet in 250 ml of ice-cold glycerol (10%). </li> | ||
− | <li>Centrifuge at | + | <li>Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant. </li> |
<li>Re-suspend the pellet in ~ 20 ml of ice-cold glycerol (10%). </li> | <li>Re-suspend the pellet in ~ 20 ml of ice-cold glycerol (10%). </li> | ||
<li>Transfer to a 30 ml sterile Oakridge tube. </li> | <li>Transfer to a 30 ml sterile Oakridge tube. </li> | ||
− | <li>Centrifuge at | + | <li>Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant. </li> |
− | <li>Re-suspend the cell pellet in a final volume of 2 ml of ice-cold 10% glycerol. The cell concentration should be about 3 x | + | <li>Re-suspend the cell pellet in a final volume of 2 ml of ice-cold 10% glycerol. The cell concentration should be about 3 x 10<sup>10</sup> cells/ml.</li> |
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
+ | <br/><div class="carregris"> | ||
+ | <h3>Electroporation of Electrocompetent <i>E. coli</i> BAP 1</h3> | ||
+ | |||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/a/a1/IGEM-Pasteur_illustration-Protocols2.jpg", height=600px /> | ||
+ | </center> | ||
− | |||
<p> | <p> | ||
<ul> | <ul> | ||
<li>Thaw the cells on ice. </li> | <li>Thaw the cells on ice. </li> | ||
− | <li>In a cold, 1.5 ml polypropylene microfuge tube, mix | + | <li>In a cold, 1.5 ml polypropylene microfuge tube, mix 40 μl of the cell suspension with 5 μl of DNA (DNA should be in a low ionic strength buffer such as TE). </li> |
<li>Mix well and incubate on ice for 1 minute.</li> | <li>Mix well and incubate on ice for 1 minute.</li> | ||
− | <li>Set the MicroPulser to “Ec1” : for 0.1 cm cuvettes U=1 | + | <li>Set the MicroPulser to “Ec1” : for 0.1 cm cuvettes U=1.8 kV and 1 pulse.</li> |
<li>Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom. </li> | <li>Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom. </li> | ||
<li>Place the cuvette in the chamber slide. </li> | <li>Place the cuvette in the chamber slide. </li> | ||
Line 595: | Line 697: | ||
<li>Quickly but gently re-suspend the cells with a Pasteur pipette. </li> | <li>Quickly but gently re-suspend the cells with a Pasteur pipette. </li> | ||
<li>Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 °C for 1 hour, shaking at 225 rpm. </li> | <li>Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 °C for 1 hour, shaking at 225 rpm. </li> | ||
− | <li>Plate | + | <li>Plate 200 μL of the cell suspension on a Petri Dish LB+ appropriate antibiotic.</li> |
<li>Incubate overnight at 37 °C. </li> | <li>Incubate overnight at 37 °C. </li> | ||
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
− | + | <br/><div class="carregris"> | |
− | + | ||
− | < | + | |
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− | + | ||
<h3>pNP-Assay</h3> | <h3>pNP-Assay</h3> | ||
<p> | <p> | ||
Line 631: | Line 709: | ||
<li>Preparation of PCS Buffer (1M, pH=7.4) </li> | <li>Preparation of PCS Buffer (1M, pH=7.4) </li> | ||
<ul> | <ul> | ||
− | <li> | + | <li>8.0 g of NaCl </li> |
− | <li>0. | + | <li>0.2 g of KCl </li> |
− | <li>1. | + | <li>1.44 g of Na<sub>2</sub>HPO<sub>4</sub> </li> |
− | <li>0. | + | <li>0.24 g of KH<sub>2</sub>PO<sub>4</sub> </li> |
<li>1L distilled H<sub>2</sub>O.</li> | <li>1L distilled H<sub>2</sub>O.</li> | ||
</ul> | </ul> | ||
− | <li>Preparation of 4- | + | <li>Preparation of 4-Nitrophenyl butyrate (pNP) in H<sub>2</sub>O. </li> |
<ul> | <ul> | ||
− | <li>Add 88µL of pNP in | + | <li>Add 88µL of pNP in 912 µL acetonitrile (ACN) (500 mM) </li> |
− | <li>Dilute to get 1ml of pNP solution at | + | <li>Dilute to get 1ml of pNP solution at 1 mM</li> |
</ul> | </ul> | ||
− | <li>Preparation of the Bacterial culture at OD( | + | <li>Preparation of the Bacterial culture at OD(600 nm)=0.1 </li> |
<ul> | <ul> | ||
− | <li>Measure OD( | + | <li>Measure OD(600 nm) of bacterial suspension. </li> |
<li>Dilute the bacterial suspension with PBS buffer. </li> | <li>Dilute the bacterial suspension with PBS buffer. </li> | ||
</ul> | </ul> | ||
− | < | + | |
− | <li>In each well of a 96-wells plate (flat-bottom), add | + | <center> |
+ | <table border="1" cellspacing="0" cellpadding="0" width="423"> | ||
+ | <tr> | ||
+ | <td width="32"><p> </p></td> | ||
+ | <td width="122"><p> </p></td> | ||
+ | <td width="95"><p align="center">NB-Esterase</p></td> | ||
+ | <td width="174"><p align="center">pNP Concentration (mM)</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32" rowspan="2"><p align="center">C1</p></td> | ||
+ | <td width="122"><p align="center">Test</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p align="center">50</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="122"><p align="center">Test</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p align="center">10</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32" rowspan="2"><p align="center">C2</p></td> | ||
+ | <td width="122"><p align="center">Test</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p align="center">50</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="122"><p align="center">Test</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p align="center">10</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32" rowspan="2"><p align="center">C3</p></td> | ||
+ | <td width="122"><p align="center">Test</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p align="center">50</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="122"><p align="center">Test</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p align="center">10</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32"><p align="center">C1</p></td> | ||
+ | <td width="122"><p align="center">Neg. control 1.a</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p> </p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32"><p align="center">C2</p></td> | ||
+ | <td width="122"><p align="center">Neg. control 1.b</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p> </p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32"><p align="center">C3</p></td> | ||
+ | <td width="122"><p align="center">Neg. control 1.c</p></td> | ||
+ | <td width="95"><p align="center">+</p></td> | ||
+ | <td width="174"><p> </p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32"><p align="center">C-</p></td> | ||
+ | <td width="122"><p align="center">Neg. control 2</p></td> | ||
+ | <td width="95"><p align="center">-</p></td> | ||
+ | <td width="174"><p align="center">50</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32"><p align="center">C-</p></td> | ||
+ | <td width="122"><p align="center">Neg. control 3</p></td> | ||
+ | <td width="95"><p align="center">-</p></td> | ||
+ | <td width="174"><p align="center">10</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="32"><p align="center">C-</p></td> | ||
+ | <td width="122"><p align="center">Neg. control 4</p></td> | ||
+ | <td width="95"><p align="center">-</p></td> | ||
+ | <td width="174"><p> </p></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </center> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | <li>In each well of a 96-wells plate (flat-bottom), add 100 µL of bacterial suspension (OD(600 nm)=0.1))</li> | ||
<li>Incubate at 34°C. </li> | <li>Incubate at 34°C. </li> | ||
− | <li>In each well, add | + | <li>In each well, add 10 µl of pNP solution. </li> |
− | <li>For | + | <li>For 30 min, OD(405 nm) is recorded every minute for each solution.</li> |
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
− | <h3> Preparation of | + | <br/><div class="carregris"> |
+ | <h3> Preparation of Electro-competent <i>Saccharomyces cerevisiae</i> cells. </h3> | ||
<p> | <p> | ||
<ul> | <ul> | ||
− | <li>Inoculate | + | <li>Inoculate 500 mL of YPD in a 2.8 L Fernbach flask with an aliquot from an overnight culture of <i>Saccharomyces cerevisiae</i>. </li> |
− | <li> Incubate at 30°C overnight, shaking at 250 | + | <li> Incubate at 30°C overnight, shaking at 250 rpm. </li> |
− | <li> Chill the cells on ice water for | + | <li> Chill the cells on ice water for 15 min to stop growth. </li> |
− | <li>Decant the cells into to sterile 250 ml centrifuge bottles and pellet the cells by centrifugation at | + | <li>Decant the cells into to sterile 250 ml centrifuge bottles and pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C.</li> |
<li>Carefully pour off and discard the supernatant ; place the centrifuge bottles with the cell pellets on ice.</li> | <li>Carefully pour off and discard the supernatant ; place the centrifuge bottles with the cell pellets on ice.</li> | ||
− | <li>Add | + | <li>Add 50 ml of sterile, ice-cold water to each of the bottles and vortex to re-suspend the cell pellets </li> |
− | <li>Bring the volume in each of the | + | <li>Bring the volume in each of the centrifuge bottles to 250 ml.</li> |
− | <li>Pellet the cells by centrifugation at | + | <li>Pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C ; pour off and discard the supernatant.</li> |
− | <li>Wash the cells with a total of | + | <li>Wash the cells with a total of 250 ml sterile, ice cold water.</li> |
− | <li> | + | <li>Re-suspend the cell pellet in 20 ml of sterile, ice cold Sorbitol(1M) and transfer to a chilled 30 ml Oakridge tube. </li> |
− | <li>Pellet the cells by centrifugation at | + | <li>Pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C</li> |
<li>Pour off and discard the supernatant.</li> | <li>Pour off and discard the supernatant.</li> | ||
− | <li> | + | <li>Re-suspend the cells pellet in 0.5 mL of sterile, ice cold sorbitol ; the final cell volume should be around 1.3 ml.</li> |
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
− | + | <br/><div class="carregris"> | |
− | <h3> Electroporation in | + | <h3> Electroporation in Electro-competent <i>Saccharomyces cerevisiae</i> cells. </h3> |
<p> | <p> | ||
<ul> | <ul> | ||
− | <li>Pipette the DNA samples (5- | + | <li>Pipette the DNA samples (5-100 ng) to be electroporated into sterile 1.5 mL microfuge tubes. Place tubes on ice. </li> |
<li>Add the competent cells:</li> | <li>Add the competent cells:</li> | ||
<ul> | <ul> | ||
− | <li>If 0.2 cm cuvettes are used, add 40 | + | <li>If 0.2 cm cuvettes are used, add 40 µl of the competent cells to each DNA sample </li> |
− | <li>If 0 | + | <li>If 0.4 cm cuvettes are used, add 80 µl of the competent cells to each DNA sample. </li> |
</ul> | </ul> | ||
− | <li>Mix gently and incubate on ice for | + | <li>Mix gently and incubate on ice for 5 min. </li> |
<li>Set the MicroPulser to « Sc2 » when using 0.2 cm cuvettes or to « Sc4 » when using 0.4 cm cuvettes. See Section 4 for operating instructions. </li> | <li>Set the MicroPulser to « Sc2 » when using 0.2 cm cuvettes or to « Sc4 » when using 0.4 cm cuvettes. See Section 4 for operating instructions. </li> | ||
− | <li>Transfer the DNA-cell samples to the appropriate electroporation | + | <li>Transfer the DNA-cell samples to the appropriate electroporation cuvettes that have been chilled on ice and tap the suspension to the bottom of the tube. </li> |
<li>Place the cuvette in the chamber slide. </li> | <li>Place the cuvette in the chamber slide. </li> | ||
<li>Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber. </li> | <li>Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber. </li> | ||
<li>Pulse once. </li> | <li>Pulse once. </li> | ||
− | <li>Remove the cuvette from the chamber and | + | <li>Remove the cuvette from the chamber and immediately add 1 ml of ice cold sorbitol (1M) to the cuvette. Gently transfer the diluted cells into a sterile tube. </li> |
− | <li>Check and | + | <li>Check and re-cold the pulse parameters. The time constant should be close to 5 milliseconds. </li> |
<li>Plate aliquots of the electroporated cells on selective agar plates containing sorbitol (1M).</li> | <li>Plate aliquots of the electroporated cells on selective agar plates containing sorbitol (1M).</li> | ||
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
− | <h3> Transformation of SK1 | + | <br/><div class="carregris"> |
+ | <h3> Transformation of SK1 yeast by electroporation</h3> | ||
<p> | <p> | ||
<ul> | <ul> | ||
<li>Inoculate 10 ml of YPD with a yeast strain and grow to saturation at 30°C with shaking.</li> | <li>Inoculate 10 ml of YPD with a yeast strain and grow to saturation at 30°C with shaking.</li> | ||
− | <li>Dilute into 40 ml of YPD in a 250 ml or larger sterile flask and grow for 2 | + | <li>Dilute into 40 ml of YPD in a 250 ml or larger sterile flask and grow for 2 hours at 30°C with shaking. </li> |
<li>Collect the cells by centrifugation at 1,000 X g for 5 min. </li> | <li>Collect the cells by centrifugation at 1,000 X g for 5 min. </li> | ||
− | <li>Decant the supernatant and | + | <li>Decant the supernatant and re-suspend the pellet in 18 ml of TE Buffer. Then add 2 ml of 1 mM lithium acetate (LiAc). </li> |
<li>Incubate cells at 30°C on a roller drum for 45 min. </li> | <li>Incubate cells at 30°C on a roller drum for 45 min. </li> | ||
− | <li>Add 500 μl of DTT (1M).</li> | + | <li>Add 500 μl of DTT (Dithiothreitol)(1M).</li> |
<li>Incubate for 15 min at 30°C on a roller drum. </li> | <li>Incubate for 15 min at 30°C on a roller drum. </li> | ||
− | <li>Add 80 ml of sterile | + | <li>Add 80 ml of sterile deionized distilled water (ddH<sub>2</sub>O) at room temperature. </li> |
<li>Centrifuge cells at 1,000 X g for 5 min. </li> | <li>Centrifuge cells at 1,000 X g for 5 min. </li> | ||
− | <li>Decant the supernatant and | + | <li>Decant the supernatant and re-suspend the pellet in 100 ml of sterile ddH2O.</li> |
<li>Again centrifuge cells at 1,000 X g for 5 min. </li> | <li>Again centrifuge cells at 1,000 X g for 5 min. </li> | ||
− | <li>Decant the supernatant and | + | <li>Decant the supernatant and re-suspend the pellet in 5 ml of 1 M Sorbitol. </li> |
<li>Centrifuge cells at 1,000 X g for 5 min. </li> | <li>Centrifuge cells at 1,000 X g for 5 min. </li> | ||
− | <li>Decant supernatant and put cells on ice. | + | <li>Decant supernatant and put cells on ice. Re-suspend the pellet in 120 μl of cold Sorbitol (1M). The volume of resuspended cells should be around 180 μl. </li> |
<li>Keep cells on ice and mix in sterile microfuge tubes </li> | <li>Keep cells on ice and mix in sterile microfuge tubes </li> | ||
<ul> | <ul> | ||
<li>40 μl of competent yeast cells </li> | <li>40 μl of competent yeast cells </li> | ||
− | <li>1.7 μl of | + | <li>1.7 μl of carrier DNA (Salmon sperm DNA)(15 mg/ml) </li> |
<li>DNA to be transformed (up to 5 μl) <li> | <li>DNA to be transformed (up to 5 μl) <li> | ||
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
− | <h3></h3> | + | <br/><div class="carregris"> |
+ | <h3>Making competent cells</h3> | ||
<p> | <p> | ||
<ul> | <ul> | ||
− | <li></li> | + | <li>Pre-grow cells in 10 ml YPglu and incubate at 30°C O/N with shaking.</li> |
− | <li></ | + | <li>Dilute to 4x10<sup>6</sup> cells/ml for <i>S. cerevisiae</i> and to 1.5 .10<sup>7</sup> cells/ml for <i>C. glabrata</i> in 50 ml YPglu and grow at 30°C with shaking for 3 to 4 h, until 2-3 .10<sup>7</sup> cells/ml for <i>S. cerevisiae</i> and ~6 .10<sup>7</sup> cells/ml for <i>C. glabrata</i> (approximately 3 generations). |
− | < | + | Rq: You will need 1 .10<sup>8</sup> cells per transformation, so you can adjust the volume of the culture if you planned to do more than 10 transformations for <i>S. cerevisiae</i> or 30 for <i>C. glabrata</i>.</li> |
− | < | + | <li>Harvest enough cells for the number of transformation planned and transfer them in a sterile tube that goes to the centrifuge: 1 .10<sup>8</sup> cells for one transformation, 2 .10<sup>8</sup> cells for 2 transformations.</li> |
− | < | + | <li>Centrifuge culture at 4°C, 5 min, 5,000 rpm.</li> |
− | <li></li> | + | <li>Wash cells with 20 ml of sterile TE/LiAc.</li> |
− | <li></li> | + | <li>Centrifuge at 4°C, 5 min at 5 000 rpm.</li> |
− | <li></li> | + | <li>Re-suspend pellet in TE/LiAc in order to have 2 x 10<sup>9</sup> c/ml : 50 µl per transformation (take into account the volume of the pellet)</li> |
− | <li></li> | + | <li>Incubate at 30°C for 15 min without shaking.</li> |
− | <li></li> | + | |
− | <li></li> | + | |
</ul> | </ul> | ||
</p> | </p> | ||
+ | </div> | ||
+ | |||
+ | <br/><div class="carregris"> | ||
+ | <h3>Transformation</h3> | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Prepare one Eppendorf tube per transformation (include a positive control and a negative control).</li> | ||
+ | <li>Add 300 µl of TE/LiAc/PEG (made the same day).</li> | ||
+ | <li>Add 50 µg of carrier DNA (5 µl) that was previously denatured for 3 min at 95°C. </li> | ||
+ | <li>Add DNA to be transformed (in 1 to 10 µl). Mix with Vortex.</li> | ||
+ | <li>Add 50 µl of competent cells per tube (10<sup>8</sup> cells/transformation) and mix carefully with pipetting up and down. </li> | ||
+ | <li>Incubate at 30°C for 30 min without shaking.</li> | ||
+ | <li>Heat shock 20 min at 42°C.</li> | ||
+ | <li>Centrifuge 5 min at 2,000 rpm.</li> | ||
+ | <li>Re-suspend in 500 µl 5 mM CaCl<sub>2</sub> and incubate at RT for 5-10 min.</li> | ||
+ | <li>Centrifuge 5 min at 2,000 rpm and re-suspend in H<sub>2</sub>O.</li> | ||
+ | <li>Spread cells with glass beads on selective media, 1/100, 1/10 or more of the transformation.</li> | ||
+ | </div> | ||
+ | |||
+ | <br> | ||
+ | <br> | ||
<!-- Renvoie haut de page --> | <!-- Renvoie haut de page --> |
Latest revision as of 21:57, 19 November 2015
![](https://static.igem.org/mediawiki/2015/c/c5/Experiments_%26_Protocols_pasteur2015.jpg)
Polymerase Chain Reaction
![](https://static.igem.org/mediawiki/2015/e/ed/IGEM-Pasteur_PCR-PreMix.jpg)
![](https://static.igem.org/mediawiki/2015/4/49/Igem-Pasteur_PCR.jpg)
1) PCR Amplification using TaKaRa Ex Taq DNA polymerase
- In a 0.2ml tube, set up the following reaction:
- Set up the following cycles in a PCR machine
- Initial denaturation: 94°C for 30 sec
- 30 cycles :
- 94°C for 30 sec
- 55°C - 65°C for 1 min depending on your annealing temperature
- 72°C 0.5-1 min per kb
- Final extension: 72°C for 5 min.
|
Tube |
Control |
Control |
Control |
Control |
Ex taq Buffer (10X) |
5 µl |
5 µl |
5 µl |
5 µl |
5 µl |
dNTP mix |
4 µl |
4 µl |
4 µl |
4 µl |
4 µl |
Template DNA |
1-10 ng |
1-10 ng |
1-10 ng |
1-10 ng |
|
Forward Primer |
final concentration: 0.2 µM |
|
final concentration: 0.2 µM |
|
final concentration: 0.2 µM |
Reverse Primer |
final concentration: 0.2 µM |
|
|
final concentration: 0.2 µM |
final concentration: 0.2 µM |
Nuclease free water |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
Ex Taq DNA Polymerase |
0.25 µl |
0.25 µl |
0.25 µl |
0.25 µl |
0.25 µl |
Total |
50 µl |
50 µl |
50 µl |
50 µl |
50 µl |
2) PCR Amplification using Phusion DNA polymerase
- In a 0.2ml tube, set up the following reaction:
- Set up the following cycles in a PCR machine
- Initial denaturation : 98°C for 30 sec
- 30 cycles :
- 94°C for 5-10s
- 45°C - 72°C for 10 to 30 sec depending on your annealing temperature
- 72°C for 15-30 sec per kb
- Final extension: 72°C for 5 min.
|
Tube |
Control |
Control |
Control |
Control |
Phusion HF Buffer (5X) |
10 µl |
10 µl |
10 µl |
10 µl |
10 µl |
dNTPs (10mM) |
1 µl |
1 µl |
1 µl |
1µl |
1 µl |
Template DNA |
<250 ng |
<250 ng |
<250 ng |
<250 ng |
|
10µM forward Primer |
2.5 µl |
|
2.5 µl |
|
2.5 µl |
10µM reverse primer |
2.5 µl |
|
|
2.5 µl |
2.5 µl |
Nuclease free water |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
Phusion DNA Polymerase |
0.5 µl |
0.5 µl |
0.5 µl |
0.5 µl |
0.5 µl |
Total |
50 µl |
50 µl |
50 µl |
50 µl |
50 µl |
3) PCR Amplification using Q5 High Fidelity Master Mix DNA polymerase
- In a 0.2ml tube, set up the following reaction:
- Set up the following cycles in a PCR machine
- Initial denaturation : 98°C for 30 sec
- 30 cycles :
- 94°C for 5-10s
- 45°C - 72°C for 10 to 30 sec depending on your annealing temperature
- 72°C for 20-30 sec per kb
- Final extension: 72°C for 2 min.
|
Tube |
Control |
Control |
Control |
Control |
Q5 HIgh Fidelity Master Mix (2X) |
25 µl |
25 µl |
25 µl |
25 µl |
25 µl |
Template DNA |
<1 ng |
<1 ng |
<1 ng |
<1 ng |
<1 ng |
10 µM forward Primer |
2.5 µl |
|
2.5 µl |
|
2.5 µl |
10 µM reverse primer |
2.5 µl |
|
|
2.5 µl |
2.5 µl |
Nuclease free water |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
Total |
50 µl |
50 µl |
50 µl |
50 µl |
50 µl |
Enzymatic Digestion
![](https://static.igem.org/mediawiki/2015/8/87/Igem-Pasteur_DigestionMix.jpg)
1) Single restriction enzyme digestion
- In a MicroCentrifuge tube, set up the following reaction on ice:
- Pipette up and down to homogenize the solution.
- Quick spin in a MicroCentrifuge (5 sec).
- Incubation at 37°C for 1h.
- Heat inactivation for 20 min at 80°C. Optional
- Add the phosphatase: Add 1 unit of Shrimp alkaline phosphatase for each pmol of phosphate end.
- Incubate for 30 min at 37°C.
- Inactivate the phosphatase at 65°C for 15 min.
|
Tube |
Control |
Buffer (10X) |
5 µl |
5 µl |
DNA |
1 µg |
1 µg |
Restriction Enzyme |
1 µl |
|
DNAse, RNAse free water |
to 50 µl |
to 50 µl |
Total |
50 µl |
50 µl |
2) Double digestion
- In a MicroCentrifuge tube, set up the following reaction on ice:
- Pipette up and down to homogenize the solution.
- Quick spin in a Micro-centrifuge (5 sec).
- Incubation at 37°C for 1h.
- Heat inactivation for 20 min at 80°C. Optional
- Add the phosphatase: Add 1 unit of Shrimp alkaline phosphatase for each pmol of phosphate end.
- Incubate for 30 min at 37°C.
- Inactivate the phosphatase at 65°C for 15 min.
|
Tube |
Control |
Control |
Control |
Control |
Buffer (10X) |
5 µl |
5 µl |
5 µl |
5 µl |
5 µl |
DNA |
1 µg |
1 µg |
1 µg |
1 µg |
0 |
Restriction Enzyme n°1 |
1 µl |
|
1 µl |
|
1 µl |
Restriction Enzyme n°2 |
1 µl |
|
|
1 µl |
1 µl |
Nuclease free water |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
to 50 µl |
Total |
50 µl |
50 µl |
50 µl |
50 µl |
50 µl |
Agarose Gel Electrophoresis
![](https://static.igem.org/mediawiki/2015/a/a8/IGEM-Pasteur_illustration-Protocols.jpg)
- Prepare the different dilutions of TAE Buffer (50X, 1X, 0.5X)
- Agarose gel preparation :
- Weigh the agarose (Invitrogen Ref 16500-500) depending on the size of your DNA.
- Dissolve it in the appropriate amount of TAE Buffer (1X).
- Heat the preparation in the microwave until the agarose is dissolved.
- Cool the preparation by letting cool water flow against the Erlenmeyer until there is no evaporation.
- Add 1 drop of EB (Eurobio Ref GEPBET02-AF) under the extraction hood. Mix gently.
- Pour the solution in the casting tray and remove any bubbles.
- Place the combs in the casting tray and let it rest until the gel is solid.
- Carefully remove the combs from the gel
- Place the agarose gel in the electrophoresis apparatus filled with TAE buffer (0.5X).
- Gel loading:
- Load 2 µl of DNA ladder in the first and eventually the last well.
- Load each well with the appropriate amount of DNA and loading buffer.
- Close the electrophoresis unit and run the gel for 1 hour at 130 V and 7 mA.
QIAgen Gel Extraction Kit Protocol
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
- Weigh the gel slice in a colorless tube
- Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 μl). For >2% agarose gels, add 6 volumes Buffer QG.
- Incubate at 50°C for until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel.
- Check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
- Add 1 gel volume of isopropanol to the sample and mix.
- Place a QIAquick spin column in a provided 2 ml collection tube.
- Apply the sample to the QIAquick column and centrifuge for 1 min.
- Discard flow-through and place the QIAquick column back into the same tube.
- To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 1 min
- Discard flow-through and place the QIAquick column back into the same tube.
- Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 17,900 x g to remove residual wash buffer.
- Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min.
- Let the column stand for 1 min,
- Centrifuge for 1 min.
- If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
![](https://static.igem.org/mediawiki/2015/2/21/IGEM_Pasteur_Gel_extraction_kit.jpg)
Ligation
![](https://static.igem.org/mediawiki/2015/e/e3/Igem-Pasteur_LigationMix.jpg)
- Thaw the T4 DNA Ligase Buffer and DNA at room temperature.
- Set up the following reaction in a microcentrifuge tube on ice :
- Gently mix the reaction by pipetting up and down
- Quick spin in a MicroCentrifuge (5 sec).
- For cohesive ends, incubation at 16°C for 1h.
- Heat inactivation for 10min at 65°C.
|
Tube |
T4 DNA ligase Buffer (10X) |
2µl |
Vector DNA |
|
Insert DNA |
|
T4 DNA ligase |
1µL |
Nuclease free water |
to 20 µl |
Total |
20µl |
Transformation in chemically competent E. coli DH5-α
![](https://static.igem.org/mediawiki/2015/8/84/Igem-Pasteur_TransformationMix.jpg)
- Thaw out on ice one tube of chemically competent E. coli DH5-α.
- Place 50µL of cells in a pre-chilled Micro-centrifuge tube.
- Refreeze any unused cells.
- Add 1-10ng of DNA to the cells and mix gently by tapping on the tube. Do not mix by pipetting up and down.
- Incubate on ice for 30 min.
- Heat shock the cells for 40 sec in a 42°C water bath.
- Place the tubes on ice for 3 minutes.
- Add 700 µl of pre-warmed (37°C) SOC medium (Invitrogen NO 15544-034)
- Incubate at 37°C for 40 min at 200 rpm.
- Spread 200 µl of transformed cells on the appropriate medium.
- Incubate overnight at 37°C
Stab Cultures
- Prepare and autoclave 0.7% LB agar (standard LB medium containing 7 g/liter agar).
- Cool the LB agar to below 50°C (when you can hold it comfortably) and add the appropriate antibiotic(s). While still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify.
- Using a sterile straight wire, pick a single colony from a freshly grown plate and stab it deep down into the soft agar several times.
- Incubate the vial at 37°C for 8–12 h leaving the cap slightly loose.
- Seal the vial tightly and store in the dark, preferably at 4°C.
MiniPrep
1) Bacterial Culture Growth
- Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic.
- Incubate for 8 h at 37°C with vigorous shaking (~300 rpm).
2) Plasmid purification
- Dilute the starter culture 1/500 to 1/1000 into selective LB medium.
- Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).
- Centrifuge at 6,000 x g for 15 min at 4°C.
- Re-suspend the bacterial pellet in 4 ml Buffer P1.
- Add 4 ml Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times
- Incubate at room temperature (15–25°C) for 5 min.
- Add 4 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times.
- Incubate on ice for 15 min.
- Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant containing plasmid DNA promptly.
- Centrifuge the supernatant again at ≥20,000 x g for 15 min at 4°C. Remove supernatant containing plasmid DNA promptly.
- Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
- Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
- Wash the QIAGEN-tip with 2 x 10 ml Buffer QC.
- Elute DNA with 5 ml Buffer QF.
- Collect the eluate in a 15 ml or 50 ml tube.
- Add 3.5 ml (0.7 volumes) room-temperature isopropanol to the eluted DNA.
- Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C.
- Carefully decant the supernatant.
- Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of TE 8.1 Buffer (Tris 10 mM, EDTA 0.1 mM).
MidiPrep
1) Bacterial Culture Growth
- Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic.
- Incubate for 12–16 h at 37°C with vigorous shaking.
- Centrifugation at > 8,000 rpm (6,800 x g) in MicroCentrifuge for 3 min at room temperature (15–25°C).
- Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.
2) Plasmid purification
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a micro-centrifuge tube.
- Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
- Centrifuge for 10 min at 13,000 rpm in a MicroCentrifuge.
- Apply the supernatants to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30–60 sec. Discard the flow-through.
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 sec. Discard the flow-through.
- Add 0.75 ml Buffer PE and centrifuge for 30–60 s.
- Discard the flow-through, and centrifuge at full speed for an additional 1 min.
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.
- Add 50 μl Buffer EB (Qiagen Elution buffer) (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column.
- Let stand for 1 min.
- Centrifuge for 1 min.
Preparation of Electro-competent E. coli BAP 1.
All the following steps take place under sterile conditions and on ice
- Inoculate 500mL of L-broth with 1/100 volume of a fresh overnight E. coli BAP 1 culture.
- Grow the cells at 37 °C at 300 rpm.
- Chill cells on ice for about 20 min.
- Transfer the cells to a cold centrifuge bottle and spin at 4,000 x g for 15 minutes at 4 °C.
- Carefully pour off and discard the supernatant. It is better to sacrifice a few cells than to leave supernatant behind.
- Gently re-suspend the pellet in 500 ml of ice-cold glycerol (10%).
- Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
- Re-suspend the pellet in 250 ml of ice-cold glycerol (10%).
- Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
- Re-suspend the pellet in ~ 20 ml of ice-cold glycerol (10%).
- Transfer to a 30 ml sterile Oakridge tube.
- Centrifuge at 4,000 x g for 15 minutes at 4 °C. Carefully pour off and discard the supernatant.
- Re-suspend the cell pellet in a final volume of 2 ml of ice-cold 10% glycerol. The cell concentration should be about 3 x 1010 cells/ml.
Electroporation of Electrocompetent E. coli BAP 1
![](https://static.igem.org/mediawiki/2015/a/a1/IGEM-Pasteur_illustration-Protocols2.jpg)
- Thaw the cells on ice.
- In a cold, 1.5 ml polypropylene microfuge tube, mix 40 μl of the cell suspension with 5 μl of DNA (DNA should be in a low ionic strength buffer such as TE).
- Mix well and incubate on ice for 1 minute.
- Set the MicroPulser to “Ec1” : for 0.1 cm cuvettes U=1.8 kV and 1 pulse.
- Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the suspension to the bottom.
- Place the cuvette in the chamber slide.
- Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
- Pulse once.
- Remove the cuvette from the chamber and add 2 ml of SOC medium to the cuvette.
- Quickly but gently re-suspend the cells with a Pasteur pipette.
- Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 °C for 1 hour, shaking at 225 rpm.
- Plate 200 μL of the cell suspension on a Petri Dish LB+ appropriate antibiotic.
- Incubate overnight at 37 °C.
pNP-Assay
- Preparation of PCS Buffer (1M, pH=7.4)
- 8.0 g of NaCl
- 0.2 g of KCl
- 1.44 g of Na2HPO4
- 0.24 g of KH2PO4
- 1L distilled H2O.
- Preparation of 4-Nitrophenyl butyrate (pNP) in H2O.
- Add 88µL of pNP in 912 µL acetonitrile (ACN) (500 mM)
- Dilute to get 1ml of pNP solution at 1 mM
- Preparation of the Bacterial culture at OD(600 nm)=0.1
- Measure OD(600 nm) of bacterial suspension.
- Dilute the bacterial suspension with PBS buffer.
- In each well of a 96-wells plate (flat-bottom), add 100 µL of bacterial suspension (OD(600 nm)=0.1))
- Incubate at 34°C.
- In each well, add 10 µl of pNP solution.
- For 30 min, OD(405 nm) is recorded every minute for each solution.
|
|
NB-Esterase |
pNP Concentration (mM) |
C1 |
Test |
+ |
50 |
Test |
+ |
10 |
|
C2 |
Test |
+ |
50 |
Test |
+ |
10 |
|
C3 |
Test |
+ |
50 |
Test |
+ |
10 |
|
C1 |
Neg. control 1.a |
+ |
|
C2 |
Neg. control 1.b |
+ |
|
C3 |
Neg. control 1.c |
+ |
|
C- |
Neg. control 2 |
- |
50 |
C- |
Neg. control 3 |
- |
10 |
C- |
Neg. control 4 |
- |
|
Preparation of Electro-competent Saccharomyces cerevisiae cells.
- Inoculate 500 mL of YPD in a 2.8 L Fernbach flask with an aliquot from an overnight culture of Saccharomyces cerevisiae.
- Incubate at 30°C overnight, shaking at 250 rpm.
- Chill the cells on ice water for 15 min to stop growth.
- Decant the cells into to sterile 250 ml centrifuge bottles and pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C.
- Carefully pour off and discard the supernatant ; place the centrifuge bottles with the cell pellets on ice.
- Add 50 ml of sterile, ice-cold water to each of the bottles and vortex to re-suspend the cell pellets
- Bring the volume in each of the centrifuge bottles to 250 ml.
- Pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C ; pour off and discard the supernatant.
- Wash the cells with a total of 250 ml sterile, ice cold water.
- Re-suspend the cell pellet in 20 ml of sterile, ice cold Sorbitol(1M) and transfer to a chilled 30 ml Oakridge tube.
- Pellet the cells by centrifugation at 3,000 X g for 5 min at 4°C
- Pour off and discard the supernatant.
- Re-suspend the cells pellet in 0.5 mL of sterile, ice cold sorbitol ; the final cell volume should be around 1.3 ml.
Electroporation in Electro-competent Saccharomyces cerevisiae cells.
- Pipette the DNA samples (5-100 ng) to be electroporated into sterile 1.5 mL microfuge tubes. Place tubes on ice.
- Add the competent cells:
- If 0.2 cm cuvettes are used, add 40 µl of the competent cells to each DNA sample
- If 0.4 cm cuvettes are used, add 80 µl of the competent cells to each DNA sample.
- Mix gently and incubate on ice for 5 min.
- Set the MicroPulser to « Sc2 » when using 0.2 cm cuvettes or to « Sc4 » when using 0.4 cm cuvettes. See Section 4 for operating instructions.
- Transfer the DNA-cell samples to the appropriate electroporation cuvettes that have been chilled on ice and tap the suspension to the bottom of the tube.
- Place the cuvette in the chamber slide.
- Push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
- Pulse once.
- Remove the cuvette from the chamber and immediately add 1 ml of ice cold sorbitol (1M) to the cuvette. Gently transfer the diluted cells into a sterile tube.
- Check and re-cold the pulse parameters. The time constant should be close to 5 milliseconds.
- Plate aliquots of the electroporated cells on selective agar plates containing sorbitol (1M).
Transformation of SK1 yeast by electroporation
- Inoculate 10 ml of YPD with a yeast strain and grow to saturation at 30°C with shaking.
- Dilute into 40 ml of YPD in a 250 ml or larger sterile flask and grow for 2 hours at 30°C with shaking.
- Collect the cells by centrifugation at 1,000 X g for 5 min.
- Decant the supernatant and re-suspend the pellet in 18 ml of TE Buffer. Then add 2 ml of 1 mM lithium acetate (LiAc).
- Incubate cells at 30°C on a roller drum for 45 min.
- Add 500 μl of DTT (Dithiothreitol)(1M).
- Incubate for 15 min at 30°C on a roller drum.
- Add 80 ml of sterile deionized distilled water (ddH2O) at room temperature.
- Centrifuge cells at 1,000 X g for 5 min.
- Decant the supernatant and re-suspend the pellet in 100 ml of sterile ddH2O.
- Again centrifuge cells at 1,000 X g for 5 min.
- Decant the supernatant and re-suspend the pellet in 5 ml of 1 M Sorbitol.
- Centrifuge cells at 1,000 X g for 5 min.
- Decant supernatant and put cells on ice. Re-suspend the pellet in 120 μl of cold Sorbitol (1M). The volume of resuspended cells should be around 180 μl.
- Keep cells on ice and mix in sterile microfuge tubes
- 40 μl of competent yeast cells
- 1.7 μl of carrier DNA (Salmon sperm DNA)(15 mg/ml)
- DNA to be transformed (up to 5 μl)
Making competent cells
- Pre-grow cells in 10 ml YPglu and incubate at 30°C O/N with shaking.
- Dilute to 4x106 cells/ml for S. cerevisiae and to 1.5 .107 cells/ml for C. glabrata in 50 ml YPglu and grow at 30°C with shaking for 3 to 4 h, until 2-3 .107 cells/ml for S. cerevisiae and ~6 .107 cells/ml for C. glabrata (approximately 3 generations). Rq: You will need 1 .108 cells per transformation, so you can adjust the volume of the culture if you planned to do more than 10 transformations for S. cerevisiae or 30 for C. glabrata.
- Harvest enough cells for the number of transformation planned and transfer them in a sterile tube that goes to the centrifuge: 1 .108 cells for one transformation, 2 .108 cells for 2 transformations.
- Centrifuge culture at 4°C, 5 min, 5,000 rpm.
- Wash cells with 20 ml of sterile TE/LiAc.
- Centrifuge at 4°C, 5 min at 5 000 rpm.
- Re-suspend pellet in TE/LiAc in order to have 2 x 109 c/ml : 50 µl per transformation (take into account the volume of the pellet)
- Incubate at 30°C for 15 min without shaking.
Transformation
- Prepare one Eppendorf tube per transformation (include a positive control and a negative control).
- Add 300 µl of TE/LiAc/PEG (made the same day).
- Add 50 µg of carrier DNA (5 µl) that was previously denatured for 3 min at 95°C.
- Add DNA to be transformed (in 1 to 10 µl). Mix with Vortex.
- Add 50 µl of competent cells per tube (108 cells/transformation) and mix carefully with pipetting up and down.
- Incubate at 30°C for 30 min without shaking.
- Heat shock 20 min at 42°C.
- Centrifuge 5 min at 2,000 rpm.
- Re-suspend in 500 µl 5 mM CaCl2 and incubate at RT for 5-10 min.
- Centrifuge 5 min at 2,000 rpm and re-suspend in H2O.
- Spread cells with glass beads on selective media, 1/100, 1/10 or more of the transformation.
^
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