Toggle navigation Home Team Team Media Collaborations Sponsors Acknowledgements Project Background Design CRISPR/Cas9 Strategy Experiments Notebook Results Perspective Interlab Study Parts Parts Basic Parts Composite Parts Human Pratices Overview Societal Issues of CRISPR/Cas9 Responsible Research and Innovation GMO regulation Integrated Practices Engagement Model Attributions Safety Contents 1 Tuesday 9th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Heat shock transformation 1.1.1.2 Extraction of pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009 1.1.1.3 Phusion PCR on pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009 1.1.1.4 pUC19 digestion with HincII enzyme 1.1.1.5 pUC19 ligation with gBlocks ATG linker RFB, detection, St sgRNA, ATG linker FKBP, Nm sgRNA, 1.2 and 4.2 1.1.2 Bringing DNA closer 1.1.2.1 Preculture of DH5a cells transformed with Sp and Td dCas9 Tuesday 9th August Lab work Visualization Heat shock transformation By Charlène and Terrence For 1.2, 4.2, SgNm, SgSt1, Detection, FRB and FKBP gBlocks, the results of the sequencing were not as expected.So, we transformed pPS16_002, pPS16_008, pPS16_010, pPS16_011, pPS16_012, pPS16_013 and pPS16_014 in DH5a. We followed the usual protocol. 50µL of cells were streaked on LB + Amp (50µg/mL) + IPTG + xGal. Extraction of pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009 By Caroline Plasmids pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009 were extracted folllowing this protocol. Phusion PCR on pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009 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 used. The products were put for migration on a 0.8%agarose gel with BET. Gel 1 PCR was made again to increase PCR products quantities. gel2 pUC19 digestion with HincII enzyme By Alice 5 µL of pUC19 plasmids were digested with 5µL of tango buffer 10X, 38µL of sterile water, and 1 µL of HincII enzyme. The mix was incubated at 37°C for 1 hour. After incubation, 1µL of HincII enzyme was added again, and the mix was incubated 1 hour again. Digestion products were migrated on a gel on August 10. pUC19 ligation with gBlocks ATG linker RFB, detection, St sgRNA, ATG linker FKBP, Nm sgRNA, 1.2 and 4.2 By Alice Ligations of gBlocks with pUC19 were performed following this protocol. The final mix was incubated at 4°C overnight. Two control conditions were tested: 1µL of linearized pUC19 + 9 µL of sterile water 1µL of linearized pUC19 + 1 µL of ligase + 1µL of ligase buffer + 7µL of sterile water With this ligation, we hope to get the plasmids below: gBlock ATG linker RFB detection St sgRNA ATG linker FKBP Nm sgRNA Plasmid name pPS16_010 pPS16_011 pPS16_012 pPS16_013 pPS16_014 Bringing DNA closer Preculture of DH5a cells transformed with Sp and Td dCas9 By Léa 200mL of liquid LB medium (spectinomycin 50µg/mL) were inoculated with DH5a cells transformed with Sp or Td dCas9 from glycerol stock, in order to extract the plasmids.
By Charlène and Terrence
For 1.2, 4.2, SgNm, SgSt1, Detection, FRB and FKBP gBlocks, the results of the sequencing were not as expected.So, we transformed pPS16_002, pPS16_008, pPS16_010, pPS16_011, pPS16_012, pPS16_013 and pPS16_014 in DH5a. We followed the usual protocol. 50µL of cells were streaked on LB + Amp (50µg/mL) + IPTG + xGal.
By Caroline
Plasmids pPS16_001, pPS16_005, pPS16_006, pPS16_007 and pPS16_009 were extracted folllowing this protocol.
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 used. The products were put for migration on a 0.8%agarose gel with BET. Gel 1
PCR was made again to increase PCR products quantities. gel2
By Alice
5 µL of pUC19 plasmids were digested with 5µL of tango buffer 10X, 38µL of sterile water, and 1 µL of HincII enzyme. The mix was incubated at 37°C for 1 hour. After incubation, 1µL of HincII enzyme was added again, and the mix was incubated 1 hour again. Digestion products were migrated on a gel on August 10.
Ligations of gBlocks with pUC19 were performed following this protocol. The final mix was incubated at 4°C overnight. Two control conditions were tested:
With this ligation, we hope to get the plasmids below:
By Léa
200mL of liquid LB medium (spectinomycin 50µg/mL) were inoculated with DH5a cells transformed with Sp or Td dCas9 from glycerol stock, in order to extract the plasmids.
