Difference between revisions of "Team:Paris Saclay/Notebook/July/8"

(→‎High fidelity PCR amplification)
(→‎High fidelity PCR amplification)
Line 32: Line 32:
 
''By Caroline''
 
''By Caroline''
  
−
The clones found with a rightful product lengths were used in another PCR this time with a high fidelity enzyme Q5. That was carry out to test the PCR product sequences. A PCR was carry out using Q5<sup>®</sup> High-Fidelity 2X Master Mix and following the [[Team:Paris_Saclay/Experiments#Q5PCR|usual protocol]] with universal puc19 primers.
+
The clones 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<sup>®</sup> High-Fidelity 2X Master Mix and following the [[Team:Paris_Saclay/Experiments#Q5PCR|usual protocol]] with universal puc19 primers.
  
  

Revision as of 08:55, 13 July 2016

Tuesday 8th 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 (1152_pheoF and 1151 5_pheoR) upstream and downstream the insertion site are chosen. . PCR products expected below :

Plasmids expected band size (bp)
pPS16_004 846
pPS16_007 744

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 amplification

By Caroline

The clones 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.


Plasmids Number of clones tested Expected band size (bp)
pPS16_001 2 998
pPS16_002 3 998
pPS16_003 2 1061
pPS16_005 2 998
pPS16_006 3 998
pPS16_007 1 746