Difference between revisions of "Team:Paris Saclay/Notebook/August/10"

(→‎2.1-2.2 and 3.1-3.2 ligation)
(→‎Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011)
Line 7: Line 7:
 
''By Alice''
 
''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 [[Team:Paris_Saclay/Experiments#taqPCR|this protocol]]. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers ([[Team:Paris_Saclay/Experiments#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.<div id="August_10"></div>
+
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 [[Team:Paris_Saclay/Experiments#taqPCR|this protocol]]. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers ([[Team:Paris_Saclay/Experiments#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.<div id="August_10"></div>
  
 
PCR products expected were :
 
PCR products expected were :

Revision as of 13:08, 10 August 2016

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_010 374
pPS16_011 1020

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.





Université Paris Sud
91405 Orsay, France
Copyright © 2016 iGEM Paris Saclay. All rights reserved All inspirations encouraged.
*This wiki was conceived during long nights of work and a great coffee experience in the beautiful Orsay, France.