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 Wednesday 31th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Colony PCR of 8 clones containing fragement 1 and 2 (dCas9 Nm GFP 10) in pSB1C3 Wednesday 31th August Lab work Visualization Colony PCR of 8 clones containing fragement 1 and 2 (dCas9 Nm GFP 10) in pSB1C3 Mahnaz Transformed cells on 26th august which had been grown overnight were used for colony PCR. For that purpose, 8 clones containing dCas9 NM - GFP 10 were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C. For each clones contained in 20 μL water, 5.13 μL of the following mix were added : 2.5 µL DreamTaq Buffer 0.5 µL of dNTPs (10mM) 1 µL of each primer mix (10µM) 0.13 μl of DreamTaq Pol PCR was performed as follow: Step Temperature Time Initial denaturation 95°C 8 min 30 cycles 95°C 30 sec Tm 30 sec 72°C t Final Extension 72°C 10 min Hold 4°C infinity Primers used were: Matrix Clones containing dCas9 NM - GFP 10 in pSB1C3 Primers iPS83 and iPS84 Tm 60.2°C t 4 min 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) dCas9 NM - GFP 10 - pSB1C3 3688
Mahnaz
Transformed cells on 26th august which had been grown overnight were used for colony PCR. For that purpose, 8 clones containing dCas9 NM - GFP 10 were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
For each clones contained in 20 μL water, 5.13 μL of the following mix were added :
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 :
