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 Tuesday 19th July 1.1 Lab work 1.1.1 Biobrick characterization 1.1.1.1 Streak BL21 bacteria on LB plate 1.1.1.2 K1372001 pre-culture 1.1.2 Visualization 1.1.2.1 Ligation of gBlock 1.2, 2.2, 4.1 within pUC19 1.1.2.2 Transformation of DH5α with 1.2, 2.2, 4.1 1.1.2.3 High fidelity PCR on bacteria transformed with pPS16_005 and pPS16_006 1.1.3 Get DNA Closer 1.1.3.1 Linearization of the DS_SPcasN and DS_TDcasN plasmids 1.1.3.2 PCR amplification from DS_SPcasN and DS_TDcasN linearized plasmids and pZA21 Tuesday 19th July Lab work Biobrick characterization Streak BL21 bacteria on LB plate By Charlène BL21 from glycerol stock were streaked on LB and incubated at 37°C overnight. K1372001 pre-culture By Naiane One isolated colony of K1372001 from a petri dish was placed in 2mL of LB + 3µL of Chloramphenicol (20µg/mL). The falcon tube containing the culture was incubated at 37°C overnight. Visualization Ligation of gBlock 1.2, 2.2, 4.1 within pUC19 By Naiane & Charlène gBlock 1.2, 2.2, 4.1 were inserted in pUC19. Two controls were made : digested pUC19 with only water digested pUC19 without gBlock Transformation of DH5α with 1.2, 2.2, 4.1 By Naiane & Charlène HeatShock competent cells were transformed with 1.2, 2.2 and 4.1. Another control was made (cells without plasmid). Cells were plated on LB + Ampicillin + IPTG + Xgal : for each gBlock, one Petri dish with 50µL of cells and another with 150µL of cells for each control, 100µL of cells They were incubated at 37°C overnight. High fidelity PCR on bacteria transformed with pPS16_005 and pPS16_006 By Alice Plasmids pPS16_005 and pPS16_006 containing gBlocks 3.1 and 3.2 were sent to sequencing. Sequencing revealed that clones 1 transformed with pPS16_005 and clone 4 transformed with pPS16_006 had the good insert in their plasmid. In ordered to assembled both inserts, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. iPS83 and iPS126 primers were used. Annealing temperature was 72°C, and initial step was prolonged to 7 min. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V. PCR products expected were : Plasmids Band size (bp) pPS16_005 964 pPS16_006 964 Get DNA Closer Linearization of the DS_SPcasN and DS_TDcasN plasmids By Caroline The DS_SPcasN and DS_TDcasN plasmids were digested with Acc65I following the usual protocol in order to facilitated the PCR. PCR amplification from DS_SPcasN and DS_TDcasN linearized plasmids and pZA21 By Caroline The different parts needed for de getting DNA closer tool were amplify by high fidelity PCR using Q5 following usual protocol with a TM at 65°C for pZA21 and a TM at 66°C for the 5 first cycles and at 72°C for the last 25 ones for DS-SPcasN and DS_TDcasN. Specific primers were used to amplify. These primers will allow the Gibson assembly afterward : Ptet_R and Ptet_F for the plasmids, Link-TDdcas_F and Ter_TDdcas_R for DS_TDcasN, Tet-SPdcas_F and Link-SPdcas_R for DS_SPcasN.
By Charlène
BL21 from glycerol stock were streaked on LB and incubated at 37°C overnight.
By Naiane
One isolated colony of K1372001 from a petri dish was placed in 2mL of LB + 3µL of Chloramphenicol (20µg/mL).
The falcon tube containing the culture was incubated at 37°C overnight.
By Naiane & Charlène
gBlock 1.2, 2.2, 4.1 were inserted in pUC19. Two controls were made :
HeatShock competent cells were transformed with 1.2, 2.2 and 4.1. Another control was made (cells without plasmid). Cells were plated on LB + Ampicillin + IPTG + Xgal :
They were incubated at 37°C overnight.
By Alice
Plasmids pPS16_005 and pPS16_006 containing gBlocks 3.1 and 3.2 were sent to sequencing. Sequencing revealed that clones 1 transformed with pPS16_005 and clone 4 transformed with pPS16_006 had the good insert in their plasmid. In ordered to assembled both inserts, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. iPS83 and iPS126 primers were used. Annealing temperature was 72°C, and initial step was prolonged to 7 min. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V.
PCR products expected were :
By Caroline
The DS_SPcasN and DS_TDcasN plasmids were digested with Acc65I following the usual protocol in order to facilitated the PCR.
The different parts needed for de getting DNA closer tool were amplify by high fidelity PCR using Q5 following usual protocol with a TM at 65°C for pZA21 and a TM at 66°C for the 5 first cycles and at 72°C for the last 25 ones for DS-SPcasN and DS_TDcasN. Specific primers were used to amplify. These primers will allow the Gibson assembly afterward : Ptet_R and Ptet_F for the plasmids, Link-TDdcas_F and Ter_TDdcas_R for DS_TDcasN, Tet-SPdcas_F and Link-SPdcas_R for DS_SPcasN.
