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 Result of migration The result shows that Gibson assembly did not work out.
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 :
The result shows that Gibson assembly did not work out.
