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 Wednesday 3st August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Glycerol stocks 1.1.1.2 High fidelity Q5 PCR on pPS16_004, pPS16_006 and pPS16_007 1.1.1.3 Extraction of the plasmids containing gBlocks pPS16_001, pPS16_002, pPS16_003, FRB, sg-ST1, FKPB, DS-NMcasN-, spacer and detection 1.1.1.4 Extraction of the plasmids containing gBlocks 1.1.2 Interlab Study 1.1.2.1 Preculture of device one transformed DH5a Wednesday 3st August Lab work Visualization Glycerol stocks By Caroline The bacteria transformed with the plasmids sent the 2/08/2016 and those that would be sent the 4/08/2016 were put into glycerol. 1mL of liquid culture and 0.5mL of glycerol were put at -20°C. High fidelity Q5 PCR on pPS16_004, pPS16_006 and pPS16_007 By Caroline Q5 PCR was carried out following the usual protocol adapted to 50µL at a Tm of 72°C. The primers used were specific to amplify only the sequences of interest. The products were put for migration on a 0.8%agarose gel with BET. Only, PCR form pPS16_004 and pPS16_007 showed a positive result. Extraction of the plasmids containing gBlocks pPS16_001, pPS16_002, pPS16_003, FRB, sg-ST1, FKPB, DS-NMcasN-, spacer and detection By Laetitia The extraction was performed without kit, following the usual protocol. It was done on: Puc 19 containing Clone pPS16_001 4 pPS16_002 3 pPS16_003 2, 5 FRB 5 sg-ST1 2, 3, 5 DS-NMcasN- 3 FKBP 4 Spacer 1, 6 Detection 3, 4, 6 Extraction of the plasmids containing gBlocks By Naiane The kit "Charge Switch-Pro Plasmid Miniprep" was used to extract plasmidic DNA of the plasmid pPS16_003 containing the Gblock 2.1, plasmid pPS16_004 containing the Gblock 2.2, plasmid pPS16_006 containing the Gblock 3.2 and the plasmid pPS16_007 containing the Gblock 4.1 from 3mL of overnight culture. Plasmids were resuspended in 100μL of Milli-Q water. DNA stored at -20°C. Interlab Study Preculture of device one transformed DH5a By Lea Two colonies were inoculated inside of two tubes of 3mL of liquid LB medium containing 30 µg/ml Chloramphenicol. The tubes were incubated at 37°C overnight.
By Caroline
The bacteria transformed with the plasmids sent the 2/08/2016 and those that would be sent the 4/08/2016 were put into glycerol. 1mL of liquid culture and 0.5mL of glycerol were put at -20°C.
Q5 PCR was carried out following the usual protocol adapted to 50µL at a Tm of 72°C. The primers used were specific to amplify only the sequences of interest. The products were put for migration on a 0.8%agarose gel with BET. Only, PCR form pPS16_004 and pPS16_007 showed a positive result.
By Laetitia
The extraction was performed without kit, following the usual protocol.
It was done on:
By Naiane
The kit "Charge Switch-Pro Plasmid Miniprep" was used to extract plasmidic DNA of the plasmid pPS16_003 containing the Gblock 2.1, plasmid pPS16_004 containing the Gblock 2.2, plasmid pPS16_006 containing the Gblock 3.2 and the plasmid pPS16_007 containing the Gblock 4.1 from 3mL of overnight culture. Plasmids were resuspended in 100μL of Milli-Q water. DNA stored at -20°C.
By Lea
Two colonies were inoculated inside of two tubes of 3mL of liquid LB medium containing 30 µg/ml Chloramphenicol. The tubes were incubated at 37°C overnight.
