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 Friday 19th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Q5 PCR on fragments 3 and 4 1.1.1.2 G 1.1.1.3 Plasmids extraction 1.1.1.4 Colony and Extraction product DreamTaq PCR Friday 19th August Lab work Visualization Q5 PCR on fragments 3 and 4 "By Charlène" A Q5 PCR was made on fragments 3 and 4 for a gel extraction. Tm = 72°C. Elongation time = 1 minute. Problem : 25 cycles at the place of 30. Q5 PCR of fragments 3 and 4 G "By Terrence, Alice and Mahnaz" There is not enough product for the extraction gel : a second PCR was done with 30 cycles. It will be running on gel next Monday. Plasmids extraction "By Terrence, Alice and Mahnaz" The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit: pPS16_009 (GFP1-9) clone 4 pPS16_009 (GFP1-9) clone 5 pPS16_014 (sgRNA Nm) clone 1 pPS16_014 (sgRNA Nm) clone 2 pPS16_013 (FKBP) clone 3 pPS16_013 (FKBP) clone 4 pPS16_013 (FKBP) clone 5 pPS16_010 (FRB) clone 2 pPS16_010 (FRB) clone 3 pPS16_010 (FRB) clone 5 pPS16_002 (1.2) clone 1 pPS16_002 (1.2) clone 11 Nano drop : Plasmid name Concentration (ng/µL) 260/230 260/280 pPS16_009 (GFP1-9) clone 4 207.03 1.76 1.85 pPS16_009 (GFP1-9) clone 5 280.30 1.79 1.89 pPS16_014 (sgRNA Nm) clone 1 350.24 2.24 1.97 pPS16_014 (sgRNA Nm) clone 2 187.62 2.20 1.05 pPS16_013 (FKBP) clone 3 272.37 2.32 1.93 pPS16_013 (FKBP) clone 4 404.65 2.34 1.94 pPS16_013 (FKBP) clone 5 204.87 2.32 1.94 pPS16_010 (FRB) clone 2 256.44 2.35 1.96 pPS16_010 (FRB) clone 3 73.04 2.13 2.05 pPS16_010 (FRB) clone 5 129.86 2.16 1.87 pPS16_002 (1.2) clone 1 49.72 1.34 1.67 pPS16_002 (1.2) clone 11 392.66 2.33 1.95 Colony and Extraction product DreamTaq PCR "By Léa and Naiane" A Colony PCR was performed on Dh5a transformed with PJet containing sgRNA Nm or 1.2 gBlocks. sgRNA Nm clones 6, 7, 8, 10, 11 and 12 were screened, and 1.2 clones 13 to 18 were screened and plated on Petri dish containing solid LB and Ampicilin. In the meantime, a DreamTaq PCR was performed on plasmids extractions listed above, using the same PCR mix. The PCR mix was made following the usual protocol. Universal primers for PJET were used (Tm= 51.9°). A previous step of denaturation (95°C, 5min) was performed for colony PCR.
"By Charlène"
A Q5 PCR was made on fragments 3 and 4 for a gel extraction. Tm = 72°C. Elongation time = 1 minute. Problem : 25 cycles at the place of 30.
"By Terrence, Alice and Mahnaz"
There is not enough product for the extraction gel : a second PCR was done with 30 cycles. It will be running on gel next Monday.
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
Nano drop :
"By Léa and Naiane"
A Colony PCR was performed on Dh5a transformed with PJet containing sgRNA Nm or 1.2 gBlocks. sgRNA Nm clones 6, 7, 8, 10, 11 and 12 were screened, and 1.2 clones 13 to 18 were screened and plated on Petri dish containing solid LB and Ampicilin.
In the meantime, a DreamTaq PCR was performed on plasmids extractions listed above, using the same PCR mix. The PCR mix was made following the usual protocol. Universal primers for PJET were used (Tm= 51.9°).
A previous step of denaturation (95°C, 5min) was performed for colony PCR.
