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|K1372001 and pcl_TAA, pclTAG or pclTq 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 1.1.1.6 Gel Extraction of 1.2 and Lig 4 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 for migration 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. Result of the extraction of pPS16003, pPS16004 and GFP1-9_PSB1C3 (clones 2 and 3) and result of the migration of gBlock 2-3-4 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|K1372001 and pcl_TAA, pclTAG or pclTq By Charlène 3 clones from each transformation condition (K1372001 and pcl_TAA, pclTAG or pclTq) 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 amplify them 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 Gel Extraction of 1.2 and Lig 4 By Terrence The PCR products of gblock 1.2 and ligation 4 were purified on gel using the NucleoSpin Gel and PCR Clean-up kit from Macherey-Nagel. Gel extraction fo 1.2 and Lig 4
By Charlène
Clones 2 and 3 of GFP1-9_PSB1C3, and pPS16003, pPS16004 were extracted with the Plasmid MiniPrep kit.
The extracts were put for migration 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 (K1372001 and pcl_TAA, pclTAG or pclTq) 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 amplify them 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 :
The PCR products of gblock 1.2 and ligation 4 were purified on gel using the NucleoSpin Gel and PCR Clean-up kit from Macherey-Nagel.
