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 Thursday 3rd August 1.1 Visualization 1.1.1 Glycerol stocks 1.1.2 High fidelity Q5 PCR on pPS16_004, pPS16_006 and pPS16_007 1.1.3 Extraction of the plasmids containing gBlocks pPS16_001, pPS16_002, pPS16_003, FRB, sg-ST1, FKPB, DS-NMcasN-, spacer and detection 1.1.4 Extraction of the plasmids containing gBlocks 1.1.5 pPS16_001, pPS16_002, pPS16_009, pPS16_014 DreamTaq PCR 1.2 Interlab Study 1.2.1 Preculture of device one transformed DH5a Thursday 3rd August 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. pPS16_001, pPS16_002, pPS16_009, pPS16_014 DreamTaq PCR By Mathilde and Laetitia DreamTaq PCR was carried out following the usual protocol. Only the clone 9 for PS16_001 (1.1), the clone 11 for PS16_002 (1.2), the clone 7 for PS16_009 (GFP) and clones 7 and 11 for pPS16_014 (Nm) were right. PS16_001 (1.1) PS16_002 (1.2) PS16_009 (GFP) pPS16_014 (Nm) 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 Mathilde and Laetitia
DreamTaq PCR was carried out following the usual protocol. Only the clone 9 for PS16_001 (1.1), the clone 11 for PS16_002 (1.2), the clone 7 for PS16_009 (GFP) and clones 7 and 11 for pPS16_014 (Nm) were right.
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.
