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 25th August 1.1 Visualization 1.1.1 Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB 1.1.2 Q5 high fidelity PCR of ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004 1.1.3 Gel electrophoresis of Q5 PCR ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004 1.1.4 Ligation of pPS16_003-pPS16_004 Thursday 25th August Visualization Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB By Mahnaz Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C. For each clones contained in 20 μl water, 4.13 μL of the following mix were added : 2.5 µL DreamTaq Buffer 0.5 µL of dNTPs (10mM) 0.5 µL of each primer(10µM) 0.13 μl of DreamTaq Pol PCR was performed as follow: Step Temperature Time Initial denaturation 95°C 3 min 25 cycles 94°C 30 sec Tm 30 sec 72°C t Final Extension 72°C 5min Hold 4°C $\infinity\$ Primers used were: Matrix plasmid pJET coding sg-Nm plasmid pJET coding FRB Primers pJET R and pJET F pJET R and pJET F Tm 60.0C 60.0C t 15 sec 15 sec 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 374 sg-Nm 362 We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying Q5 high fidelity PCR of ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004 By mathilde Fragment 1 (pPS16_001-pPS16_002) and fragment 2 (pPS16_003-pPS16_004E) were each tested three times (A,B,C) according to the following protocol : 10µL of Q5 buffer 1µL of dNTPs (10mM) Primers mix (1µL each) 1µL of ligation product 0,25 µL of Q5 high fidelity polymerase 37,75 µL of nuclease free water The primers used were IPS83 and IPS 122 for the fragment 1, and IPS 84 and 123 for the fragment 2. The PCR programm featured 30s of initial denaturation, 72°c of annealing temperature for 30s and 2min of final extension. Gel electrophoresis of Q5 PCR ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004 PCR products were put to migration following the usual protocol. PCR products Expected band size (bp) pPS16_001-pPS16_002 1920 pPS16_003-pPS16_004 1831 Result of the migration Only pPS16_001-pPS16_002 ligation product shows strands at the expected size. Ligation of pPS16_003-pPS16_004 By Mathilde 4 µL of pPS16_003 plasmid 4 µL of pPS16_004 plasmid 1µL of T4 Buffer 10X 1µL of T4 Ligase The solution is put in incubation at 4°c over night.
By Mahnaz
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
For each clones contained in 20 μl water, 4.13 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 :
By mathilde
Fragment 1 (pPS16_001-pPS16_002) and fragment 2 (pPS16_003-pPS16_004E) were each tested three times (A,B,C) according to the following protocol :
The primers used were IPS83 and IPS 122 for the fragment 1, and IPS 84 and 123 for the fragment 2. The PCR programm featured 30s of initial denaturation, 72°c of annealing temperature for 30s and 2min of final extension.
PCR products were put to migration following the usual protocol.
Only pPS16_001-pPS16_002 ligation product shows strands at the expected size.
By Mathilde
The solution is put in incubation at 4°c over night.
