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 Thursday 6th October 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Colony PCR of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) Thursday 6th October Lab work Visualization Colony PCR of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) By Maxence For that purpose, X clones 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 48.4°C 30 sec 72°C 1 min Final Extension 72°C 7 min Hold 4°C $\infty$ Primers used were: Matrix Clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) Primers iPS168 and iPS169 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 - GFP 11 - FKBP - GFP 10 1714 GEL
By Maxence
For that purpose, X clones 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 :
GEL
