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 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, that containing gBlocks 1.1, 3.1 and GFP 1-9 respectively, were sent to sequencing. Sequencing revealed that clones 2 transformed with pPS16_001, clone 1 transformed with pPS16_005, and clone 1 transformed with pPS16_009 had the good insert in their plasmid. In ordered to assembled gBlocks, a 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, and initial step was prolonged to 7 min. 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_005 3.1 iPS138 iPS126 960 pPS16_009 GFP 1-9 iPS138 iPS139 862 No PCR products were expected. We supposed that initial denaturation was too long, and caused enzyme damages. By 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, that containing gBlocks 1.1, 3.1 and GFP 1-9 respectively, were sent to sequencing. Sequencing revealed that clones 2 transformed with pPS16_001, clone 1 transformed with pPS16_005, and clone 1 transformed with pPS16_009 had the good insert in their plasmid. In ordered to assembled gBlocks, a 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, and initial step was prolonged to 7 min. 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 :
No PCR products were expected. We supposed that initial denaturation was too long, and caused enzyme damages.
By
