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 Glycerol stocks of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 (pPS16_018) 1.1.1.2 Plasmids extraction of clones 7, 8, 9 and 10 containing GFP 1.9 in pSB1C3 (pPS16_018) 1.1.1.3 PCR of pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 to correct mutations 1.1.1.4 Gel of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 Monday 19th September Lab work Visualization Glycerol stocks of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 (pPS16_018) "By Maxence, Mahnaz, Coline & Caroline" The glycerol stock of the bacteria with the following plasmids were made. pPS16_018 (FKBP - GFP 10) clone 7 pPS16_018 (FKBP - GFP 10) clone 8 pPS16_018 (FKBP - GFP 10) clone 9 pPS16_018 (FKBP - GFP 10) clone 10 500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C. Plasmids extraction of clones 7, 8, 9 and 10 containing GFP 1.9 in pSB1C3 (pPS16_018) "By Maxence, Mahnaz Coline & Caroline" The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit: pPS16_018 (FKBP - GFP 10) clone 7 pPS16_018 (FKBP - GFP 10) clone 8 pPS16_018 (FKBP - GFP 10) clone 9 pPS16_018 (FKBP - GFP 10) clone 10 PCR of pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 to correct mutations By Maxence, Mahnaz, Coline & Caroline 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 (pPS16_017) clone 8 dCas9 ST - GFP 11 (pPS16_017) clone 8 Primers iPS174 and iPS175 iPS173 and iPS176 Tm 72°C 72°C t 3min 50sec Gel of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 By Maxence, Mahnaz, Coline & Caroline 4 µL of each PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min. PCR products expected were : PCR products Expected band size (bp) Product 1 obtained by iPS174 & iPS175 5837 Product 2 obtained by iPS173 & iPS176 1599 GEL
"By Maxence, Mahnaz, Coline & Caroline"
The glycerol stock of the bacteria with the following plasmids were made.
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
"By Maxence, Mahnaz Coline & Caroline"
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
By Maxence, Mahnaz, Coline & Caroline
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:
4 µL of each PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
GEL
