Difference between revisions of "Team:Paris Saclay/Notebook/June/28"
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=Tuesday 28th June= | =Tuesday 28th June= | ||
==Lab work== | ==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=== | ===Interlab study=== | ||
Revision as of 14:54, 4 July 2016
Template:Team: Paris Saclay/notebook header
Contents
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
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.
| 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 controls were made :
- 1µL digested vector + 9µL H2O
- 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL H2O
- 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL insert
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.
