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 27th September 1.1 Lab work 1.1.1 Visualization 1.1.1.1 PCR of extracted GFP 1.9 in pSB1C3 (pPS16_020) 1.1.1.2 Gel of PCR products Tuesday 27th September Lab work Visualization PCR of extracted GFP 1.9 in pSB1C3 (pPS16_020) By Maxence & Mahnaz In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids sent for sequencing the 14th September (clones 2, 7, 8 and 12) and plasmids extracted the 23rd Sepembter (clones 3, 4, 7 and 8) were used. For each 50μl of reaction, mix the following reagents : 1 µL of matrix 1 µL of dNTPs (10mM) 2.5 µL of each primer mix (10µM) 10 µL of buffer (5X) 0,5 µL of Phusion polymerase 32.5 µL of nuclease free water Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow: Step Temperature Time Initial denaturation 95°C 30sec 30 cycles 95°C 30sec 64.4°C 30sec 72°C 30sec Final Extension 72°C 5min Hold 4°C $\infty$ Primers used were: Matrix Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020) Primers iPS168 and iPS169 Gel of PCR products By Maxence & Manhaz 4 µL of PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity. PCR products expected were : PCR products Expected band size (bp) GFP 1.9 1135 GEL
By Maxence & Mahnaz
In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids sent for sequencing the 14th September (clones 2, 7, 8 and 12) and plasmids extracted the 23rd Sepembter (clones 3, 4, 7 and 8) were used.
For each 50μl of reaction, mix the following reagents :
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:
Primers used were:
By Maxence & Manhaz
4 µL of PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.
PCR products expected were :
GEL
