Tuesday 28^th June

Lab work

Stock solutions preparation

• 200 mL 60% glycerol stock: 120mL glycerol 100% + 80mL water.
• 200mL CH₃COOK 5M stock: 98.15g of CH₃COOK powder + 60mL water, shake and add water to 200mL
• 100mL Solution III (plasmid extraction protocol): 28.5 mL water + 60mL CH₃COOK 5M + 11.5 mL of pure liquid CH₃COOH
• 200mL TE stock: 2mL Tris+ 0.4 mL EDTA + water to 200mL

Interlab study

Cytometer

By Caroline, Alice, Lea and Charlene, with Isabelle help

Cells culture created on the 27/06/2016 were analysed with cytometer. Results were as expected.

Visualization

pUC19 digestion

TODO

pUC19 was digested as expected.

pUC19-gBlocks ligation

By Charlene

GBlocks were resuspended into 100µL of TE buffer (except for 4-1 which was resuspended into 50µL of TE) in order to get a concentration of 10ng/mL after a quick spin. Solutions were vortexed, incubated for 20min at 50°C, vortexed another time and quickly spun down. The ratio vector size/insert size x 3.5 is calculated to determine if insert is enough in excess compared to vector (it has to be superior than 7). 3.5 represents the ratio insert concentration/ vector concentration.

3 conditions were tested (2 controls):

1. 1µL digested vector + 9µL water
2. 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL water
3. 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL insert

By using ligation, we hope to obtain the following plasmids:

Transformation with ligation products

By Caroline and Charlene

DH5α cells were transformed with ligation products using the same protocol as on 24/06/2016 Transformations were displayed on Petri dishes with LB medium containing 50µg/mL of ampicillin and covered with 0.5µL of 80mg/mL XGal and 0.5µL of 1M IPTG.

Bringing DNA closer

Plasmid extraction

By Naiane and Lea

Plasmids from cell culture of the 27/06/2016 were extracted following the usual protocol.

Cells transformation

By Naiane

Extracted plasmids (2µL) were transformed into DH5α competent cells (50µL) using the usual protocol.

BioBrick characterization

Plasmid extraction

By Lea

The same protocol as on 24/06/2016 was used to extract K1372001 from DH5α cells, except the phenol chloroform step was not done.

BL21 competent cell culture

By Caroline

A colony of BL21 cells was put into 4mL of LB medium and incubated overnight at 37°C, 180 rpm.