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 17th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Extraction of pPS16003, pPS16004 and GFP1-9_PSB1C3 (clones 2 and 3) 1.1.1.2 Purification of gBlocks 2, 3 and 4 1.1.1.3 Cloning with pJET the gBlock 1.2, FRB, FKBP and SgNM 1.1.1.4 Culture of BL21 1.1.1.5 Q5 PCR on gBlocks (1.2, NM_Sg_RNA, FRB and FKBP) and products of 4.1 and 4.2 gBlocks ligation Wednesday 17th August Lab work Visualization Extraction of pPS16003, pPS16004 and GFP1-9_PSB1C3 (clones 2 and 3) By Charlène Clones 2 and 3 of GFP1-9_PSB1C3, and pPS16003, pPS16004 were extracted with the Plasmid MiniPrep kit. The extracts were put to migrated on a 0.8%agarose gel with BET. Purification of gBlocks 2, 3 and 4 By Terrence The purification was carried out following the usual protocol. Cloning with pJET the gBlock 1.2, FRB, FKBP and SgNM By Léa and Naiane The cloning was carried out using a new protocol which uses pJET as cloning vector. A heat shock transformation was made on the cloning samples using the following protocol Culture of BL21 By Charlène 3 clones from each transformation condition were grown in 500µL of LB, streptomycin (50µg/mL) and chloramphenicol (30µg/mL). Then each solution was split into 3x150µL and 350µL of mix of LB + streptomycin (50µg/mL) + chloramphenicol (30µg/mL) were added to each culture. Salicylate (main solution at 10mM) was added to obtain the following concentrations : 0, 0.03mM and 1mM. Q5 PCR on gBlocks (1.2, NM_Sg_RNA, FRB and FKBP) and products of 4.1 and 4.2 gBlocks ligation By Alice Q5 PCR was performed directly on gBlocks to try to amplified it following this protocol. Primers used were: gBlocks 1.2 NM_Sg_RNA FRB FKBP 4.1 and 4.2 gBlocks ligation Primers iPS121 and iPS122 iPS133 and iPS83 iPS149 and iPS150 iPS145 and iPS146 iPS129 and iPS84 Annealing temperature was 70°C. Elongation step was up to 1 min. After amplification, 1 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. PCR products expected were : gBlocks expected band size (bp) 1.2 960 NM_Sg_RNA 362 FRB 473 FKBP 419 4.1 and 4.2 gBlocks ligation 1994 Migration of gBlocks and ligation
By Charlène
Clones 2 and 3 of GFP1-9_PSB1C3, and pPS16003, pPS16004 were extracted with the Plasmid MiniPrep kit.
The extracts were put to migrated on a 0.8%agarose gel with BET.
By Terrence
The purification was carried out following the usual protocol.
By Léa and Naiane
The cloning was carried out using a new protocol which uses pJET as cloning vector.
A heat shock transformation was made on the cloning samples using the following protocol
3 clones from each transformation condition were grown in 500µL of LB, streptomycin (50µg/mL) and chloramphenicol (30µg/mL). Then each solution was split into 3x150µL and 350µL of mix of LB + streptomycin (50µg/mL) + chloramphenicol (30µg/mL) were added to each culture. Salicylate (main solution at 10mM) was added to obtain the following concentrations : 0, 0.03mM and 1mM.
By Alice
Q5 PCR was performed directly on gBlocks to try to amplified it following this protocol. Primers used were:
Annealing temperature was 70°C. Elongation step was up to 1 min. After amplification, 1 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
PCR products expected were :
