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.

gBlock	1-1	1-2	2-1	3-1	3-2	4-1	4-2	GFP1-9
Size (bp)	960	960	1023	960	960	706	1288	862
vector size/insert size x 3.5	9.84	9.84	9.24	9.84	9.84	13.39	7.34	10.96

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

Thus, the following plasmids were engineered:

gBlock	1-1	1-2	2-1	3-1	3-2	4-1	4-2	GFP1-9
Plasmid name	pPS16_001	pPS16_002	pPS16_003	pPS16_005	pPS16_006	pPS16_007	pPS16_008	pPS16_009

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 same protocol as on 24/06/2016.

Cells transformation

By Naiane

Extracted plasmids (2µL) were transformed into DH5α competent cells (50µL) using the same protocol as on 24/06/2016.

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.