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 11th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Transformation of DH5a cells with pPS16_009 1.1.1.2 pPS16_010 extraction 1.1.1.3 High Fidelity Phusion PCR of transformed cell with ligation 2 and 3 1.1.1.4 DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC 19 1.1.1.5 Phusion PCR on PSB1C3 Thursday 11th August Lab work Visualization Transformation of DH5a cells with pPS16_009 By Léa Dh5a cells were transformed with pPS16_009, or psB1c3 (control), or not transformed (control)using the usual protocol. pPS16_010 extraction By Alice After screening colony screening PCR, 3 clones (clones 3, 7 and 8) transformed with pPS16_010 seemed to have the insert of interest. Plasmids from these clones were extracted following this protocol. After extraction, plasmids were migrated on a gel. Band size expected: Plasmid name Plasmid size (bp) pPS16_010 3070 Migration of FRB plasmid Nano Drop: Plasmid name Concentration (ng/µL) 260/230 260/280 pPS16_010 clone 3 334.48 2.35 1.94 pPS16_010 clone 7 321.06 2.32 1.98 pPS16_010 clone 8 139.13 2.10 1.88 High Fidelity Phusion PCR of transformed cell with ligation 2 and 3 By Laetitia PCR Phusion was performed on productct of ligation containing ligation 2 and 3 (gblocks) from 09/08/16. It was done following this protocol. For ligation 2 the primers used were IPS 123 and IPS 84. For ligation 3 the primers used were IPS 128 and IPS 83. DreamTaq PCR of transformed cell with pPS16_002,008,010, 011, 012, 013, 014 and puC 19 By Léa and Laetitia PCR DreamTaq was performed on 6 clones of each transformed celles from 10/08/16. Each clone was plated and put in liquid culture with LB and Ampicillin. No PCR products were observed on the gel. Phusion PCR on PSB1C3 By Alice PSB1C3 was amplified with Phusion DNA polymerase following this protocol. Two primers (iPS41 and iPS42) were chosen. Annealing temperature was 70.9°C. Elongation step lasted 1min. After amplification, 5 µL of each PCR products with loading dye and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. PCR products expected: Plasmids expected band size (bp) PSB1C3 2070 Migration of PSB1C3 amplification
By Léa
Dh5a cells were transformed with pPS16_009, or psB1c3 (control), or not transformed (control)using the usual protocol.
By Alice
After screening colony screening PCR, 3 clones (clones 3, 7 and 8) transformed with pPS16_010 seemed to have the insert of interest. Plasmids from these clones were extracted following this protocol. After extraction, plasmids were migrated on a gel.
Band size expected:
Nano Drop:
By Laetitia
PCR Phusion was performed on productct of ligation containing ligation 2 and 3 (gblocks) from 09/08/16. It was done following this protocol. For ligation 2 the primers used were IPS 123 and IPS 84. For ligation 3 the primers used were IPS 128 and IPS 83.
By Léa and Laetitia
PCR DreamTaq was performed on 6 clones of each transformed celles from 10/08/16. Each clone was plated and put in liquid culture with LB and Ampicillin. No PCR products were observed on the gel.
PSB1C3 was amplified with Phusion DNA polymerase following this protocol. Two primers (iPS41 and iPS42) were chosen. Annealing temperature was 70.9°C. Elongation step lasted 1min. After amplification, 5 µL of each PCR products with loading dye and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
PCR products expected:
