Difference between revisions of "Team:Paris Saclay/Notebook/August/10"
(→Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011) |
(→Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011) |
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==Lab work== | ==Lab work== | ||
===Visualization=== | ===Visualization=== | ||
| + | ====Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011==== | ||
| + | ''By Alice'' | ||
| + | |||
| + | After transformation, only white bacteria are selected (blue white screen). For bacteria transformed with pPS_011, the only clone white is chosen, and for bacteria transformed with pPS16_010, 9 white clones among others are chosen. They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following [[Team:Paris_Saclay/Experiments#taqPCR|this protocol]]. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers ([[Team:Paris_Saclay/Experiments#primers|1151_pheoR and 1152_pheoF]]) upstream and downstream the insertion site are chosen. Annealing temperature was 53°C. After amplification, 1 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. This gel is also used to migrate pUC19 plamids digested with HincII.<div id="August_10"></div> | ||
| + | |||
| + | PCR products expected were : | ||
| + | |||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !Plasmids | ||
| + | !expected band size (bp) | ||
| + | |- | ||
| + | |pUC19 digested with HincII | ||
| + | |2696 | ||
| + | |- | ||
| + | |pPS16_010 | ||
| + | |374 | ||
| + | |- | ||
| + | |pPS16_011 | ||
| + | |1020 | ||
| + | |} | ||
| + | |||
==== PCR Clean-up with the NucleoSpin kit ==== | ==== PCR Clean-up with the NucleoSpin kit ==== | ||
''By Caroline'' | ''By Caroline'' | ||
Revision as of 12:57, 10 August 2016
