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 26th September 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018) 1.1.1.2 NanoDrop Measurements Monday 26th September Lab work Visualization Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018) By Maxence, Mahnaz & Coline A colony PCR was done for 16 clones (8 clones obtained by Gibson with 2 fragments and 8 clones obtained by Gibson with 3 fragments) from the 23rd September. For that purpose, 16 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 30sec Final Extension 72°C 7 min Hold 4°C $\infty$ Primers used were: Matrix Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19) 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) FKBP - GFP 10 in pSB1C3 (pPS16_019) 757 GEL NanoDrop Measurements By Maxence, Manhaz & Caroline Sample Concentration (ng/µL) GFP 1.9 in pSB1C3 (pPS16_020) clone 3 8 GFP 1.9 in pSB1C3 (pPS16_020) clone 4 14.5 GFP 1.9 in pSB1C3 (pPS16_020) clone 7 21.5 GFP 1.9 in pSB1C3 (pPS16_020) clone 8 13 PCR product containing FKBP - GFP 10 from the 22nd September 38.19 Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 2 235 Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 7 51 Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 8 200
By Maxence, Mahnaz & Coline
A colony PCR was done for 16 clones (8 clones obtained by Gibson with 2 fragments and 8 clones obtained by Gibson with 3 fragments) from the 23rd September. For that purpose, 16 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
By Maxence, Manhaz & Caroline
