Difference between revisions of "Team:Paris Saclay/Notebook/August/10"
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= Wednesday 10<sup>th</sup> August= | = Wednesday 10<sup>th</sup> August= | ||
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===Visualization=== | ===Visualization=== | ||
====Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011==== | ====Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011==== | ||
''By Alice'' | ''By Alice'' | ||
| − | After transformation, only white bacteria | + | After transformation, only white bacteria were selected (blue/white screen). For bacteria transformed with pPS16_011, the only clone white was chosen, and for bacteria transformed with pPS16_010, 9 white clones among others were chosen. PCR with DreamTaq 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]]) were used. Annealing temperature was 53°C. After amplification, 1 µL of each PCR products and 10 µL of DNA ladder were placed on gel 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 products expected were : | PCR products expected were : | ||
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| − | [[File:T--Paris_Saclay--20160810_PCR_det_FRB.jpeg| | + | [[File:T--Paris_Saclay--20160810_PCR_det_FRB.jpeg|500px|thumb|right|Migration of pPS16_010 and pPS16_011]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br> |
| − | For pPS16_010 we expected a band at | + | For pPS16_010 we expected a band at 0.4kB. We can not see on this first gel, so 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.<br> |
| − | [[File:T--Paris_Saclay-- | + | [[File:T--Paris_Saclay--20160810_PCR_det_1_FRB_3_5_7_8_1.jpeg|400px|thumb|right|Migration of pPS16_010 (extracted from clones 3,5,7 and 8) and pPS16_011 (clone 1), 20 min]] |
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| + | [[File:T--Paris_Saclay--20160810_PCR_det_1_FRB_3_5_7_8_25_1.jpeg|400px|thumb|right|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 ==== | ==== PCR Clean-up with the NucleoSpin kit ==== | ||
''By Caroline'' | ''By Caroline'' | ||
| − | The purification was carried out on PCR 1.1, 3.1, 3.2, 4.1 and GFP following the usual [[Team:Paris_Saclay/Experiments#Purification|protocol]]. They were | + | The purification was carried out on PCR 1.1, 3.1, 3.2, 4.1 and GFP following the usual [[Team:Paris_Saclay/Experiments#Purification|protocol]]. They were migrated on 0.8% agarose gel containing BET. |
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| + | [[File:T--Paris_Saclay--160810_gel_purification_PCR2.jpg|400px|thumb|right|Result of the PCR]] | ||
==== 2.1-2.2 and 3.1-3.2 ligation ==== | ==== 2.1-2.2 and 3.1-3.2 ligation ==== | ||
''By Charlène'' | ''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. | + | 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 ==== | ==== GFP and PSB1C3 digestion ==== | ||
''By Charlène'' | ''By Charlène'' | ||
| − | 8µL of GFP purify PCR products or 8µL of | + | 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 ==== | + | ==== Phusion PCR on the ligation products (2.1-2.2 and 3.1-3.2)==== |
''By Caroline'' | ''By Caroline'' | ||
| − | The PCR was carried out following the usual [[Team:Paris_Saclay/Experiments#phusion|protocol]] adapted to 50µL and with a TM at 72°C. The specific [[Team:Paris_Saclay/Experiments#primers|primers]] for each parts were | + | The PCR was carried out following the usual [[Team:Paris_Saclay/Experiments#phusion|protocol]] adapted to 50µL and with a TM at 72°C. The specific [[Team:Paris_Saclay/Experiments#primers|primers]] for each parts were used. The products were put for migration on a 0.8%agarose gel with BET. |
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| + | [[File:T--Paris_Saclay--160810_gel_PCR_phusion_ligation.jpg|400px|thumb|right|Result of the PCR]] | ||
====Transformation of ligation products and pUC 19==== | ====Transformation of ligation products and pUC 19==== | ||
''By Laetitia'' | ''By Laetitia'' | ||
| − | Heat choc | + | Heat choc transformations were 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 [[Team:Paris_Saclay/Experiments#Polymerase_chain_reaction|protocol]]. |
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| + | Each transformation product was spread on a meduim of LB, AMpicillin IPTG and xGal and put at 37°C Overnight. | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 16:34, 9 October 2016
