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 Tuesday 26th July 1.1 Lab work 1.1.1 Visualization 1.1.1.1 High fidelity PCR on bacteria transformed with pPS16_001, pPS16_003, pPS16_005 and pPS16_009 1.1.1.2 Low Fidelity Dreamtaq PCR of DH5α|pPS16_002 1.1.1.3 Tuesday 26th July Lab work Visualization High fidelity PCR on bacteria transformed with pPS16_001, pPS16_003, pPS16_005 and pPS16_009 By Alice PCR peformed on July 25 with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. Specific primers were used for each plasmids (table below). Annealing temperature calculated was 72°C. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V. PCR products expected were : Plasmids gBlocks Primer Forward Primer Reverse Band size (bp) pPS16_001 1.1 iPS138 iPS120 960 pPS16_003 2.1 iPS123 iPS124 1023 pPS16_005 3.1 iPS138 iPS126 960 pPS16_009 GFP 1-9 iPS138 iPS139 862 Low Fidelity Dreamtaq PCR of DH5α|pPS16_002 By Laetitia We made a DreamTaq PCR on DH5α|pPS16_002 transformed cultures containing the Gblock 1.2. The PCR was done on 6 clones of transformed cells: 4 clones of the Petri dish from 19/07 and 2 clones of the Petri dish from 22/07 The usual protocol was used with TM at 57°C. By
By Alice
PCR peformed on July 25 with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. Specific primers were used for each plasmids (table below). Annealing temperature calculated was 72°C. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V.
PCR products expected were :
By Laetitia
We made a DreamTaq PCR on DH5α|pPS16_002 transformed cultures containing the Gblock 1.2. The PCR was done on 6 clones of transformed cells: 4 clones of the Petri dish from 19/07 and 2 clones of the Petri dish from 22/07 The usual protocol was used with TM at 57°C.
By
