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 25th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Extraction of plasmids containing sgRNA Nm, fragment 3 + 4 (from the HIfi assembly mix) , GFP1-9 1.1.1.2 Digestion of PSB1C3 containing the fragment 3 + 4 assembly and PSB1C3 with GFP1-9's insert. 1.1.1.3 Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation Thursday 25th August Lab work Visualization Extraction of plasmids containing sgRNA Nm, fragment 3 + 4 (from the HIfi assembly mix) , GFP1-9 By Terrence The extraction was carried out following the usual protocol. The plasmid PSB1C3 with the insert : - SgRNA Nm were extracted from the clone 5 - Fragment 3 + 4 were extracted from the clone 6 - GFP1-9 were extracted from the clone 4 Digestion of PSB1C3 containing the fragment 3 + 4 assembly and PSB1C3 with GFP1-9's insert. By Terrence Extracted plasmids were digest with XbaI and PstI, following usual protocol. Result of the digestion Q5 PCR on products of 1.1 and 1.2, and of 2.1 and 2.2 gBlocks ligation By Mathilde Q5 PCR was performed directly on gBlocks to amplify them following the protocol: Prepare enough PCR master mix for two sets of triplicats analyzed plus one extra. For each 50μl of reaction, mix the following reagents : • 1µL of ligation product • 1µ of dNTPs (10mM) • 1µL of each primer mix (10µM) • 10µL of q5 buffer • 0,25µL of Q5 high fidelity polymerase • 35,75µL of nuclease free water Mix gently and aliquot 50μl of the mix into the PCR tubes on ice. Perform PCR as follow: Step Temperature Time Initial denaturation 98°C 30sec 30 cycles 98°C 5sec 72°c 30sec 72°C 1min Final Extension 72°C 2min Hold 4°C $\infty$ Primers used were: gBlocks 1.1 and 1.2 gBlocks ligation 2.1 and 2.2 gBlocks ligation Primers iPS83 and iPS122 iPS184 and iPS123 After amplification, 3 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. PCR products expected were : gBlocks expected band size (bp) 1.1 and 1.2 gBlocks ligation 960 4.1 and 4.2 gBlocks ligation 1994 Migration of gBlocks and ligation
By Terrence
The extraction was carried out following the usual protocol.
The plasmid PSB1C3 with the insert : - SgRNA Nm were extracted from the clone 5 - Fragment 3 + 4 were extracted from the clone 6 - GFP1-9 were extracted from the clone 4
Extracted plasmids were digest with XbaI and PstI, following usual protocol.
By Mathilde
Q5 PCR was performed directly on gBlocks to amplify them following the protocol:
• 1µL of ligation product • 1µ of dNTPs (10mM) • 1µL of each primer mix (10µM) • 10µL of q5 buffer • 0,25µL of Q5 high fidelity polymerase • 35,75µL of nuclease free water
Primers used were:
After amplification, 3 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
PCR products expected were :
