Tuesday 28th 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):
- 1µL digested vector + 9µL water
- 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL water
- 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL insert
By using ligation, we hope to obtain the following plasmids:
| 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
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 same protocol as on 24/06/2016.
BioBrick characterization
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.
