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 29th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 PCR of the plasmid extraction from clonies 4,5,9,12 containing FRB sequence in pJET plasmid 1.1.1.2 Samples preparation for sequencing 1.1.1.3 NanoDrop Measurements Monday 29th August Lab work Visualization PCR of the plasmid extraction from clonies 4,5,9,12 containing FRB sequence in pJET plasmid By Mahnaz 2.5 µL DreamTaq Buffer 0.5 µL of dNTPs (10mM) 1 µL of each primer (10µM) primer pJET R and F 0.13 μl of DreamTaq Pol 20 µl H2O PCR was performed as follow: Step Temperature Time Initial denaturation 95°C 3 min 30 cycles 95°C 30 sec 52 30 sec 72°C 1 min Final Extension 72°C 7min Hold 4°C \infinity\ Primers used were: Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3 Primers pJET R and F Tm 60°C t 1 min 30 After amplification, 3 µL of each PCR products and 5 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) FRB in pJET 474 Result of migration The expected size was 474 pb and the 4 plasmids were sent to sequencing. Séquençage Samples preparation for sequencing by Mahnaz 15 µL of plasmides plasmids coding FRB from colonies 4,5,9 and 12 were sent to be sequenced. 20 µL of the primers pJET F (5µM) and pJET R (5µM) were sent for sequencing. NanoDrop Measurements By Mahnaz Sample Concentration (ng/µL) PCR fragment FRB clone 4 58.43 PCR fragment FRB clone 5 320.75 PCR fragment FRB clone 9 830.99 PCR fragment FRB clone 12 473.37
By Mahnaz
PCR was performed as follow:
Primers used were:
After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
The expected size was 474 pb and the 4 plasmids were sent to sequencing.
Séquençage
by Mahnaz
15 µL of plasmides plasmids coding FRB from colonies 4,5,9 and 12 were sent to be sequenced. 20 µL of the primers pJET F (5µM) and pJET R (5µM) were sent for sequencing.
