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

(→‎Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011)
(→‎Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011)
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After transformation, only white bacteria were selected (blue and white screen).  
 
After transformation, only white bacteria were selected (blue and white screen).  
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For bacteria transformed with pPS_011, the only clone white was chosen, and for bacteria transformed with pPS16_010, 9 white clones among others were chosen. They are expected to have plasmids with the wanted inserts.
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For bacteria transformed with pPS_011, the only clone white was chosen, and for bacteria transformed with pPS16_010, 9 white clones among others were chosen. They are expected to have plasmids with the wanted inserts. PCR with DreamTaq DNA Polymerase was performed following [[Team:Paris_Saclay/Experiments#taqPCR|this protocol]]. Clones selected for PCR were 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 were 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 was also used to migrate pUC19 plasmids digested with HincII.<div id="August_10"></div>
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PCR with DreamTaq DNA Polymerase was performed following [[Team:Paris_Saclay/Experiments#taqPCR|this protocol]]. Clones selected for PCR were 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 were 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 was also used to migrate pUC19 plasmids digested with HincII.<div id="August_10"></div>
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Revision as of 13:47, 17 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 were selected (blue and white screen).

For bacteria transformed with pPS_011, the only clone white was chosen, and for bacteria transformed with pPS16_010, 9 white clones among others were chosen. They are expected to have plasmids with the wanted inserts. PCR with DreamTaq DNA Polymerase was performed following this protocol. Clones selected for PCR were kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1151_pheoR and 1152_pheoF) upstream and downstream the insertion site were 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 was also used to migrate pUC19 plasmids 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), 20 min
Migration of pPS16_010 (extracted from clones 3,5,7 and 8) and pPS16_011 (clone 1), 30 min

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. 4µL of 3.1 purify PCR products, 4µL of 3.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed. They were 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_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009(clone 7)

By Terrence

The extraction was carried out following the usual protocol.

Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009 (clone 7)


Nano Drop:

Plasmid name Concentration (ng/µL) 260/230 260/280
pPS16_011 clone 3 34.08 1.54 1.57
pPS16_011 clone 4 368.42 2.36 1.92
pPS16_011 clone 6 295.99 2.29 1.91
pPS16_014 clone 11 529.17 2.39 1.91
pPS16_002 clone 7 30.50 1.76 2.13
pPS16_009 clone 7 282.1 2.31 1.93

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.

Extraction of DS-SPcasN- and DS-TDcasN-



Nano Drop:

Plasmid name Concentration (ng/µL) 260/230 260/280
DS-SPcasN- 185.46 2.23 1.79
DS-TDcasN 198.97 1.88 1.79





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