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+ [[File:T--Paris_Saclay--160711_visualization2_échelle.jpg|400px|thumb|right|pPS16_004 (2.2) and pPS16_007 (4.1) Migration]]
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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.
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.
Revision as of 14:35, 19 July 2016
Friday 8th July Lab work Bringing DNA closer TODO
Migration of SP, SP1, SP2, NM, ST1, TD
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. After amplification, 5 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
PCR products expected were :
Plasmids
expected band size (bp)
pPS16_004
865
pPS16_007
763
pPS16_004 (2.2) and pPS16_007 (4.1) Migration
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 .
pPS16_001 (1.1), pPS16_002 (1.2) and pPS16_003 (2.1) Migration
pPS16_001, pPS16_003, pPS16_005 and pPS16_006 Migration
pPS16_004 (2.2) and pPS16_007 (4.1) Migration
