Difference between revisions of "Team:Paris Bettencourt/Notebook/Riboswitch"
Line 865: | Line 865: | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | |||
+ | <h2>Results</h2> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/f/ff/999000.png" width="350px"> | ||
+ | Nothing : ? <br> | ||
+ | 350 bp : recircularization (expected : 320 bp ) <br> | ||
+ | Expected = 1700 ! —> nothing <br> | ||
+ | |||
+ | <br><br> | ||
+ | <h1 class="date two">August 13th</h1> | ||
+ | |||
+ | <h2>Goal 1 </h2> | ||
+ | Digestion | ||
+ | |||
+ | <h2>Procedure</h2> | ||
+ | I will digest F1* this time and try to insert it in LP2* digested too | ||
+ | <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*d</th> | ||
+ | <th class="tg-031e">LP2*d</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">ADN</td> | ||
+ | <td class="tg-031e">2 ug (60,6 ng/uL) = 35 uL</td> | ||
+ | <td class="tg-031e">2 ug (108,3 ng/uL) = 20 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">EcorI</td> | ||
+ | <td class="tg-031e">4 uL</td> | ||
+ | <td class="tg-031e">4 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">PstI</td> | ||
+ | <td class="tg-031e">4 uL</td> | ||
+ | <td class="tg-031e">4 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">FastAP</td> | ||
+ | <td class="tg-031e">-</td> | ||
+ | <td class="tg-031e">4 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Buffer</td> | ||
+ | <td class="tg-031e">8 uL</td> | ||
+ | <td class="tg-031e">12 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</td> | ||
+ | <td class="tg-031e">29 uL</td> | ||
+ | <td class="tg-031e">76uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">80 uL</td> | ||
+ | <td class="tg-031e">120 uL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Then incubated 1 h at 37°C | ||
+ | |||
+ | <h2>Goal 2 </h2> | ||
+ | Ligation | ||
+ | |||
+ | <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">L4</th> | ||
+ | <th class="tg-031e">LC (control, no insert)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Insert</td> | ||
+ | <td class="tg-031e">195 ng (35 ng/uL) = 6 uL</td> | ||
+ | <td class="tg-031e">-</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Vector</td> | ||
+ | <td class="tg-031e">100 ng (35ng/uL) = 3uL</td> | ||
+ | <td class="tg-031e">3 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Ligase</td> | ||
+ | <td class="tg-031e">1 uL</td> | ||
+ | <td class="tg-031e">1 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Ligase Buffer</td> | ||
+ | <td class="tg-031e">2 uL</td> | ||
+ | <td class="tg-031e">2 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">Water</td> | ||
+ | <td class="tg-031e">8 uL</td> | ||
+ | <td class="tg-031e">14 uL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="tg-031e">TOTAL</td> | ||
+ | <td class="tg-031e">20 uL</td> | ||
+ | <td class="tg-031e">20 uL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Then waited 40 min on the bench <br> | ||
+ | And transformed 2uL of each tube in 50 uL of chemically competent cells.<br> | ||
+ | Then plated 3 petri dish (Cm 25ng/uL : 100 uL of L4; 150 uL of L4; 100 uL of LC<br> | ||
+ | 37°C overnight.<br> | ||
+ | |||
+ | <h2>Result</h2> | ||
+ | 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 |
Revision as of 18:34, 14 September 2015