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 1.1.3 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. 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. 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 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. 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.
