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 Monday 19th September 1.1 Lab work 1.1.1 Visualization 1.1.1.1 PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 to correct mutations Monday 19th September Lab work Visualization PCR of pSB1C3 from dCas9 ST - GFP 11 clone 8 to correct mutations By Maxence In order to correct mutations in dCas9 ST - GFP 11, PCR was performed with the following protocol. 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 HF (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 98°C 30sec 30 cycles 98°C 10sec 72°C 30sec 72°C t Final Extension 72°C 5min Hold 4°C $\infty$ Primers used were: Matrix dCas9 ST - GFP 11 clone 8 dCas9 ST - GFP 11 clone 8 Primers iPS174 and iPS175 iPS173 and iPS176 Tm 72°C 72°C t 3min 50sec
By Maxence
In order to correct mutations in dCas9 ST - GFP 11, PCR was performed with the following protocol.
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:
