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
|
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)
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
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 and 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
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.
Extraction of pPS16_014(clone 3 and 11), pPS16_002 (clone 7), pPS16_009(clone 7) and pPS16_011 (clone 3, 4, 6)
By Terrence
The extraction was carried out following the usual protocol.
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.
Bringing DNA closer
Extraction of DS-SPcasN- and DS-TDcasN-
By Terrence
The extraction was carried out with the Nucleobond Ax Kit.
