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 Friday 16th September Visualization Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019) By Mahnaz Colonies were obtained for the Gibson done the 15th September. A colony PCR was done for 16 clones from the 15th September. For that purpose, 16 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 3 min 30 cycles 95°C 30 sec 61°C 30 sec 72°C 30 sec Final Extension 72°C 7 min Hold 4°C $\infinity\$ Primers used were: Matrix Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19) Primers iPS83 and iPS84 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) FKBP - GFP 10 in pSB1C3 (pPS16_019) 757 Result of the migration Some PCR products were at the good size. Clones 7, 8, 9 and 10 were selected and were grown at 37°C overnight.
By Mahnaz
Colonies were obtained for the Gibson done the 15th September. A colony PCR was done for 16 clones from the 15th September. For that purpose, 16 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 :
Some PCR products were at the good size. Clones 7, 8, 9 and 10 were selected and were grown at 37°C overnight.
