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 Glycerol stocks 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 There is not enough product for the extraction gel : a second PCR was done with 30 cycles. It will be run on gel next Monday. Glycerol stocks "By Terrence, Alice and Mahnaz" The glycerol stock of the bacteria with the following plasmids were made. 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 500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. 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 The extracted plasmids were also run on agarose gel for confirmation. 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. Gel electrophoresis of colony PCR products 1.2 gBlock. Only the sample from the clone number 14 seems to have an appropriate size (expected at 1073 bp). This clone will be grown and the plasmid will be extracted to be sequenced. Gel electrophoresis of PCR products FRB, FKBP and sgRNA Nm gBlocks. For FRB (492bp), plasmid extractions from clones 5 and 2 seem to have an appropriate size and will be send for sequencing. For FKBP (537bp), the 3 plasmids extraction will be sent. For 1.2 (1073bp), only plasmid extraction from clone 11 will be sent, and no plasmid extraction will be sent for sgRNA Nm (480bp). Gel electrophoresis of colony PCR products, gBlock sg RNA Nm. No significative stripe is observed. there is no clone carrying the plasmid of interrest.
"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.
There is not enough product for the extraction gel : a second PCR was done with 30 cycles. It will be run on gel next Monday.
"By Terrence, Alice and Mahnaz"
The glycerol stock of the bacteria with the following plasmids were made.
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
Nano drop :
The extracted plasmids were also run on agarose gel for confirmation.
"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.
