Difference between revisions of "Team:Paris Bettencourt/Notebook/Riboswitch"
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Number of colonies : 100 uL of L4 = 200 ; 150 uL of L4 = 300 ; 100 uL of LC = 3 | Number of colonies : 100 uL of L4 = 200 ; 150 uL of L4 = 300 ; 100 uL of LC = 3 | ||
The control is showing that my T4 colonies might have insertions | The control is showing that my T4 colonies might have insertions | ||
+ | |||
+ | <h2>Goal 3 </h2> | ||
+ | 4 colony PCR on 24 colonies from L2 done yesterday with the control | ||
+ | |||
+ | <h2>Procedure</h2> | ||
+ | Made 4 tubes with 1 mL of water and introduced 6 colonies per tube in order to be able to do more screening. Let the tubes on the bench 1 hour (osmotic shock), then centrifugation 5 min 14 K rpm. Then took 8 uL of each suspension and put it in 4 PCR tubes. | ||
+ | <style type="text/css"> | ||
+ | .tg {border-collapse:collapse;border-spacing:0;} | ||
+ | .tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;} | ||
+ | .tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;} | ||
+ | </style> | ||
+ | <table class="tg"> | ||
+ | <tr> | ||
+ | <th class="tg-031e"></th> | ||
+ | <th class="tg-031e">PCR1</th> | ||
+ | <th class="tg-031e">PCR2</th> | ||
+ | <th class="tg-031e">PCR3</th> | ||
+ | <th class="tg-031e">C</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Colonies</td> | ||
+ | <td class="tg-031e">1-6 ;8 uL</td> | ||
+ | <td class="tg-031e">7-12 ; 8 uL</td> | ||
+ | <td class="tg-031e">13-18 ; 8 uL</td> | ||
+ | <td class="tg-031e">19-24 ; 8 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Master Mix</td> | ||
+ | <td class="tg-031e">10 uL</td> | ||
+ | <td class="tg-031e">10 uL</td> | ||
+ | <td class="tg-031e">10 uL</td> | ||
+ | <td class="tg-031e">10 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Primers 148 - 149</td> | ||
+ | <td class="tg-031e">1 uL x 2</td> | ||
+ | <td class="tg-031e">1 uL x 2</td> | ||
+ | <td class="tg-031e">1 uL x 2</td> | ||
+ | <td class="tg-031e">1 uL x 2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</td> | ||
+ | <td class="tg-031e">6 uL</td> | ||
+ | <td class="tg-031e">6 uL</td> | ||
+ | <td class="tg-031e">6 uL</td> | ||
+ | <td class="tg-031e">6 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">20 uL</td> | ||
+ | <td class="tg-031e">20 uL</td> | ||
+ | <td class="tg-031e">20 uL</td> | ||
+ | <td class="tg-031e">20 uL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Result = nothing on the electrophoresis migration.<br> | ||
+ | It might be a problem in the protocol, maybe 1mL is to much and it’s over diluted so not enough template ?<br> | ||
+ | Maybe the cells didn’t exploded ?<br> | ||
+ | |||
+ | <br><br> | ||
+ | <h1 class="date two">August 14th</h1> | ||
+ | |||
+ | <h2>Goal </h2> | ||
+ | Colony PCR (16 colonies from T4) | ||
+ | |||
+ | <h2>Procedure</h2> | ||
+ | <style type="text/css"> | ||
+ | .tg {border-collapse:collapse;border-spacing:0;} | ||
+ | .tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;} | ||
+ | .tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;} | ||
+ | </style> | ||
+ | <table class="tg"> | ||
+ | <tr> | ||
+ | <th class="tg-031e">Suspended T4 colony in 20 uL of water</th> | ||
+ | <th class="tg-031e">6 uL</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Primers 148 - 149</td> | ||
+ | <td class="tg-031e">1 uL x2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Master Mix Phusion</td> | ||
+ | <td class="tg-031e">8 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">16 uL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | x 16<br> | ||
+ | Anealing 51°C<br> | ||
+ | Elongation time : 1:30 min<br> | ||
+ | 35 cycles<br> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/c/cc/5656.png" width="350px"> | ||
+ | Selected colony 6 for overnight culture in LB broth + Cm25<br> | ||
+ | Also colony 5 for a control<br> | ||
+ | The electrophoresis shows a bad level of insertion ( It is strange considering that my control - had just 3 colonies growing )<br> | ||
+ | Why 2 bands in the 6th colony ? Maybe multiple plasmid insertion some with insertion, some without.<br> | ||
+ | What to do?<br> | ||
+ | Work on the 6th colony<br> | ||
+ | Change my restriction enzymes<br> | ||
+ | Use DpnI<br> |
Revision as of 16:07, 15 September 2015