Friday 8^th July

Lab work

Visualization

Colony screening PCR

on bacteria transformed with pPS16_004 and pPS16_007

By Alice

After transformation, only white bacteria are selected (blue white screen). 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. PCR products expected were :

5 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. No PCR products are observed, due to an error in the protocol : MilliQ water was used instead of steril water. The same PCR will be performed on 11/07/16.

Electrophoresis of the screening PCR products

By Caroline

Amplification products from the 07/07/2016 screening PCR were put in a 0.8% agarose gel containing BET. The products in solution were mixed with purple loading dye and migrated for 30min. Some clones showed PCR products with lengths close to those expected.

High fidelity PCR

By Caroline

The clones (from 05/07/2016) found with a rightful product lengths were used in another PCR this time with a high fidelity enzyme Q5. We used a high fidelity enzyme because to reduce the error ratio and sequence the PCR products. A PCR was carry out using Q5^® High-Fidelity 2X Master Mix and following the usual protocol with universal pUC19 primers 1151_pheoR and 1152_pheoF.