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Team:Evry/Notebook/Transposons/16.10.14
From 2014.igem.org
(Difference between revisions)
| Line 24: | Line 24: | ||
find enzyme with a good cut numbers in the genome and not in our insert: HindIII | find enzyme with a good cut numbers in the genome and not in our insert: HindIII | ||
| - | <br><br>Then religate | + | Digestion of 8 <i>Pseudovibrio denitrificans</i> DNA preparation by HindIII |
| + | <br>50ul final volume, 200 ng DNA preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul Q5 enzyme. 1h | ||
| + | |||
| + | <br><br>Then religate: | ||
| + | <br>Ligation: | ||
<br><br>then light digestion with XbaI | <br><br>then light digestion with XbaI | ||
| + | <br>50ul final volume, 20 ul DNA religated preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul Q5 enzyme. 15 min | ||
| + | <br>deactivation at 80 C° for 20 min | ||
<br><br>then PCR | <br><br>then PCR | ||
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</p> | </p> | ||
| - | <span class="cd-date"> | + | <span class="cd-date">Oct 16</span> |
</div> | </div> | ||
</div> | </div> | ||
</html> | </html> | ||
Revision as of 22:27, 17 October 2014
Show BBa_1413044 work even empty in DH5alpha
8 colonies of DH5alpha were transformed with BBa_1413044 and grown in 3 ml LB + 2 negative control
Preparation for PCR colony:
Spin down 500 ul culture at 14000 g - 2 min, resuspend in 100 ul H20 MilliQ, put at 95 C° for 10 min then spin down at 14000 g - 2 min.
PCR: enzyme: Q5; template: 3 ul of supernatent for the culture; oligo: F 100/ R 101; Tm tested: 55; elongation time: 1m00s
defining where the transposase integrate
find enzyme with a good cut numbers in the genome and not in our insert: HindIII Digestion of 8 Pseudovibrio denitrificans DNA preparation by HindIII
50ul final volume, 200 ng DNA preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul Q5 enzyme. 1h
Then religate:
Ligation:
then light digestion with XbaI
50ul final volume, 20 ul DNA religated preparation, 5 ul Neb 2.1 buffer, 38 ul H2O, 1 ul Q5 enzyme. 15 min
deactivation at 80 C° for 20 min
then PCR
Oct 16
