Tuesday 19^th 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.