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 were selected (blue and white screen). For bacteria transformed with pPS_011, the only clone white was chosen, and for bacteria transformed with pPS16_010, 9 white clones among others were chosen. They are expected to have plasmids with the wanted inserts. PCR with DreamTaq DNA Polymerase was performed following
this protocol. Clones selected for PCR were kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (
1151_pheoR and 1152_pheoF) upstream and downstream the insertion site were 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 was also used to migrate pUC19 plasmids digested with HincII.
PCR products expected were :
| Plasmids
|
expected band size (bp)
|
| pUC19 digested with HincII
|
2696
|
| pPS16_010
|
431
|
| pPS16_011
|
1077
|
Migration of pPS16_010 and pPS16_011
For pPS16_010 we expected a band at about 0.4kB, that we can not see on this first gel, that is why we migrated again PCR products from clones 3,5,7 and 8 for bacteria transformed with pPS16_010 and clone 1 for bacteria transformed with pPS16_011.
Migration of pPS16_010 (extracted from clones 3,5,7 and 8) and pPS16_011 (clone 1), 20 min
Migration of pPS16_010 (extracted from clones 3,5,7 and 8) and pPS16_011 (clone 1), 30 min
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 migrated on 0.8% agarose gel containing BET.
gel1
2.1-2.2 and 3.1-3.2 ligation
By Charlène
4µL of 2.1 purify PCR products, 4µL of 2.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed.
4µL of 3.1 purify PCR products, 4µL of 3.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed.
They were incubated for 1h at RT.
GFP and PSB1C3 digestion
By Charlène
8µL of GFP purify PCR products or 8µL of PSB1C3, 1µL of Buffer, 0.5µL of EcoRI and 0.5µL of PstI were mixed and incubated for 1h at 37°C.
Phusion PCR on the ligation products (2.1-2.2 and 3.1-3.2)
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 used. The products were put for migration on a 0.8%agarose gel with BET.
gel2
Transformation of ligation products and pUC 19
By Laetitia
Heat choc transformation was performed on DH5a with the products of ligation (containing 1.2, 4.2, ATG link FRB, ATG linf FKBP, ST sg RNA, NM sg RNA and detection) following the usual protocol.
Each transformation product was plated on a meduim of LB, AMpicillin IPTG and Xgal and put at 37°C Overnight.
