Difference between revisions of "Team:Paris Bettencourt/Notebook/Riboswitch"
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<img src="https://static.igem.org/mediawiki/2015/1/11/7665.png" width="350px"><br> | <img src="https://static.igem.org/mediawiki/2015/1/11/7665.png" width="350px"><br> | ||
So bad | So bad | ||
+ | |||
+ | <a name="september" class="anchor"><h1></h1></a> | ||
+ | <h1 class="date three">September 1st</h1> | ||
+ | |||
+ | <h2>Goal 1</h2> | ||
+ | PCR FINAL for a new transformation | ||
+ | <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"></th> | ||
+ | <th class="tg-031e">F1</th> | ||
+ | <th class="tg-031e">F2</th> | ||
+ | <th class="tg-031e">F3</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Template</td> | ||
+ | <td class="tg-031e">2ng</td> | ||
+ | <td class="tg-031e">5ng</td> | ||
+ | <td class="tg-031e">10ng</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Primers 150-151</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">MasterMix</td> | ||
+ | <td class="tg-031e">25uL</td> | ||
+ | <td class="tg-031e">25 uL</td> | ||
+ | <td class="tg-031e">25 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</td> | ||
+ | <td class="tg-031e">19 uL</td> | ||
+ | <td class="tg-031e">16uL</td> | ||
+ | <td class="tg-031e">11uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | T°C gradient : 0 - 59°C<br> | ||
+ | 1 - 58.8°C<br> | ||
+ | 2 - 58.4°C<br> | ||
+ | 3 - 57.9°C<br> | ||
+ | 4 - 57.2°C<br> | ||
+ | 5- 56.6°C<br> | ||
+ | <h2>Result</h2> | ||
+ | Gel order : GR1KB-10-11-12-13-14-15- GR1KB-20-21-22-23-24-25- GR1KB-30-31-32-33-34-35<br> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/a/ae/2131.png" width="600px"> | ||
+ | <h2>Goal 2</h2> | ||
+ | Gibson 1 - New PCR | ||
+ | <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"></th> | ||
+ | <th class="tg-031e">B5</th> | ||
+ | <th class="tg-031e">B6</th> | ||
+ | <th class="tg-031e">B7</th> | ||
+ | <th class="tg-031e">I3</th> | ||
+ | <th class="tg-031e">I4</th> | ||
+ | <th class="tg-031e">I5</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Template</td> | ||
+ | <td class="tg-031e">1ng</td> | ||
+ | <td class="tg-031e">3ng</td> | ||
+ | <td class="tg-031e">6ng</td> | ||
+ | <td class="tg-031e">1ng</td> | ||
+ | <td class="tg-031e">3ng</td> | ||
+ | <td class="tg-031e">6ng</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Primers</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">MasterMixPhusion</td> | ||
+ | <td class="tg-031e">25uL</td> | ||
+ | <td class="tg-031e">25uL</td> | ||
+ | <td class="tg-031e">25uL</td> | ||
+ | <td class="tg-031e">25uL</td> | ||
+ | <td class="tg-031e">25uL</td> | ||
+ | <td class="tg-031e">25uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</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> | ||
+ | <td class="tg-031e">20 uL</td> | ||
+ | <td class="tg-031e">20 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOT</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Primers used</td> | ||
+ | <td class="tg-031e">211-212</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e">209-210</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">T°C annealing</td> | ||
+ | <td class="tg-031e">69°C</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e">66°C</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Elongation time</td> | ||
+ | <td class="tg-031e">2min</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e">15sec</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <h2>Results</h2> | ||
+ | Gel order : - - - B5 B6 B7 I4 I5 ( I3 was lost )<br> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/c/c9/7687.png" width="300px"> | ||
+ | |||
+ | |||
+ | <h1 class="date three">September 2nd</h1> | ||
+ | |||
+ | <h2>Goal 1</h2> | ||
+ | Digestion/Ligation | ||
+ | |||
+ | <h2>Procedure</h2> | ||
+ | The PCR product was purified before. <br> | ||
+ | Digestion : <br> | ||
+ | <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">F11d</th> | ||
+ | <th class="tg-031e">pSB1C3d</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">DNA 1:3</td> | ||
+ | <td class="tg-031e">55ng/uL, 1.5 ug —> 30 uL</td> | ||
+ | <td class="tg-031e">96 ng/uL, 0.75 ug —> 8 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Fast AP</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">EcoRI</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">SpeI</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">FD Buffer</td> | ||
+ | <td class="tg-031e">4uL</td> | ||
+ | <td class="tg-031e">6uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</td> | ||
+ | <td class="tg-031e">2uL</td> | ||
+ | <td class="tg-031e">40uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">40uL</td> | ||
+ | <td class="tg-031e">60uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Nanodrop after purif</td> | ||
+ | <td class="tg-031e">8.6</td> | ||
+ | <td class="tg-031e">11 (after gel extract)</td> | ||
+ | </tr> | ||
+ | </table><br> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2015/a/ab/9877.png" width="300px"><br> | ||
+ | I extracted the 3kb band from the gel | ||
+ | <br> | ||
+ | Ligation : <br> | ||
+ | <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">La (F2d’-LP1d’)</th> | ||
+ | <th class="tg-031e">La c</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">F11d</td> | ||
+ | <td class="tg-031e">25 uL</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">pSB1C3d</td> | ||
+ | <td class="tg-031e">10uL</td> | ||
+ | <td class="tg-031e">10uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Ligase</td> | ||
+ | <td class="tg-031e">1uL</td> | ||
+ | <td class="tg-031e">1uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">L buffer</td> | ||
+ | <td class="tg-031e">4uL</td> | ||
+ | <td class="tg-031e">4uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e">25uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">40uL</td> | ||
+ | <td class="tg-031e">40uL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Then on the bench for 1 hour | ||
+ | |||
+ | <h2>Goal 2</h2> | ||
+ | Back to my Gibson 1 | ||
+ | <h2>Procedure</h2> | ||
+ | ~ 60 ng of backbone in ~ 5 uL for assembly so concentrations as low as 12 ng/uL are usually fine. <br> | ||
+ | <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">G11</th> | ||
+ | <th class="tg-031e">G12</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">B5</td> | ||
+ | <td class="tg-031e">100ng — > 3uL</td> | ||
+ | <td class="tg-031e">100ng — > 3uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">I5</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | <td class="tg-031e">7.7ng (equimolar) —> 1uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">I2</td> | ||
+ | <td class="tg-031e">7.7ng (equimolar according to NEB Bio Calculator) —> 1uL</td> | ||
+ | <td class="tg-031e"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">GibsonMix</td> | ||
+ | <td class="tg-031e">15uL</td> | ||
+ | <td class="tg-031e">15uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</td> | ||
+ | <td class="tg-031e">1 uL</td> | ||
+ | <td class="tg-031e">1 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">20uL</td> | ||
+ | <td class="tg-031e">20uL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Then 60 min to 50°C<br> | ||
+ | |||
+ | Then I did a transformation by Heat Shock using 2 uL of the product and I plated it. | ||
+ | |||
+ | <h1 class="date three">September 3rd</h1> | ||
+ | |||
+ | <h2>Result</h2> | ||
+ | Result of my plating from yesterday<br> | ||
+ | G11: 200uL —> 300 colonies (3 were red)<br> | ||
+ | 20 uL —> 40 colonies (2 red)<br> | ||
+ | G12 : 200uL —> 30 colonies (9 were red)<br> | ||
+ | 20uL —> 3 colonies (1 red)<br> | ||
+ | |||
+ | <h2>Goal 1</h2> | ||
+ | Colony PCR (on colonies that don’t show any red fluo) | ||
+ | <h2>Procedure</h2> | ||
+ | 16 colonies : | ||
+ | Master Mix dream Taq 10uL , Primers Fwd 148 1uL , Primer Rv 149 1uL, Water 8uL , TOTAL 20uL<br> | ||
+ | 1 min 45 elongation expected length 1566; T°Cannealing 52°C; 95°C 8min first | ||
+ | <h2>Result</h2> | ||
+ | Colony 1 to 16, GR 1kb between 8 and 9<br> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/6/67/8977.png" width="600px"><br> | ||
+ | Liquid culture in M9 (without B12) with Cm 25 for colonies 3, 4, 5, 7, 8, 9 , 10 | ||
+ | |||
+ | <h2>Goal 2</h2> | ||
+ | Colony PCR on the red colonies | ||
+ | <h2>Procedure</h2> | ||
+ | Master Mix dream Taq 10uL , Primers Fwd 148 1uL , Primer Rv 149 1uL, Water 8uL , TOTAL 20uL<br> | ||
+ | <h2>Result</h2><br> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/9/93/8676.png" width="600px"><br> | ||
+ | Colony 11’ is quiet interesting, the 3' too<br> | ||
+ | Liquid culture in M9 Cm25 of 1’, 2’, 3’, 11’ without B12 and with 5 mg/L of adenosylcobalamin and in LB Cm25 | ||
+ | |||
+ | <h1 class="date three">September 4th</h1> | ||
+ | <h2>Goal 1</h2> | ||
+ | PCR my new G-block for Gibson 2 | ||
+ | |||
+ | <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"></th> | ||
+ | <th class="tg-031e">R (Riboswitch)</th> | ||
+ | <th class="tg-031e">B (pSB1C3)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Template DNA</td> | ||
+ | <td class="tg-031e">2ng</td> | ||
+ | <td class="tg-031e">2ng</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Primers 1 (10uM)</td> | ||
+ | <td class="tg-031e">2uL (213)</td> | ||
+ | <td class="tg-031e">2uL (215)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Primers 2 (10uM)</td> | ||
+ | <td class="tg-031e">2uL (214)</td> | ||
+ | <td class="tg-031e">2uL (216)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Phusion Master Mix</td> | ||
+ | <td class="tg-031e">25 uL</td> | ||
+ | <td class="tg-031e">25 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</td> | ||
+ | <td class="tg-031e">19 uL</td> | ||
+ | <td class="tg-031e">19 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | <td class="tg-031e">50 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">T°C annealing</td> | ||
+ | <td class="tg-031e">67°C</td> | ||
+ | <td class="tg-031e">66°C</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Time elongation</td> | ||
+ | <td class="tg-031e">25 sec</td> | ||
+ | <td class="tg-031e">1 min 10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Length</td> | ||
+ | <td class="tg-031e">476</td> | ||
+ | <td class="tg-031e">3149</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <h2>Result</h2><br> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/b/b5/%21%21%21%21%21.png" width="300px"><br> | ||
+ | Nothing |
Latest revision as of 16:04, 16 September 2015