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Line 37: ''By Caroline''
''By Caroline''
+ The PCR was carried out following the usual [[Team:Paris_Saclay/Experiments#phusion|protocol]] adapted to 50µL and with a TM at 72°C. The specific [[Team:Paris_Saclay/Experiments#primers|primers]] for each parts were using. The products were put to migrated on a 0.8%agarose gel with BET.
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Revision as of 13:00, 10 August 2016
Wednesday 10th August Lab work 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
this protocol . Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two 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.
PCR products expected were :
Plasmids
expected band size (bp)
pUC19 digested with HincII
2696
pPS16_010
374
pPS16_011
1020
PCR Clean-up with the NucleoSpin kit By Caroline
The purification was carried out on PCR 1.1, 3.1, 3.2, 4.1 and GFP following the usual protocol . They were put to migrated on 0.8% agarose gel containing BET.
2.1-2.2 and 3.1-3.2 ligation By Charlène
Phusion PCR of ligations products By Caroline
The PCR was carried out following the usual protocol adapted to 50µL and with a TM at 72°C. The specific primers for each parts were using. The products were put to migrated on a 0.8%agarose gel with BET.
