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Team:Paris Saclay/Notebook/July/22
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Step 3 - When the culture OD<sub>650</sub> = 0,6 (Step 1): | Step 3 - When the culture OD<sub>650</sub> = 0,6 (Step 1): | ||
*put in ice during 10min. | *put in ice during 10min. | ||
| - | *centriguge 4°C, 5min, 4000rpm | + | *centriguge 4°C, 5min, 4000rpm. |
| - | *Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% '''COLD''' | + | *Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% '''COLD'''. |
| + | *centriguge 4°C, 5min, 4000rpm. | ||
| + | *Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% '''COLD'''. | ||
| + | *centriguge 4°C, 5min, 4000rpm. | ||
| + | *Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% '''COLD'''. | ||
| + | |||
| + | Step 4 - Transformation: | ||
| + | *control 50µl (without DNA) | ||
| + | *50µl transformed bacteria + 2,5µl plasmid + 7,5µl pOSV 230 PCR (purifié) | ||
| + | |||
| + | Step 5 - Electroporation control | ||
| + | |||
| + | |||
'''Members there''': | '''Members there''': | ||
Revision as of 20:34, 23 July 2014
Contents |
Tuesday 22st July
Lab Work
1 - Extraction of p cola plasmid DNA
by Sean
p cola is a low-copy plasmid, and thus requires a slightly different protocol than other plasmids.
2 - Samples for stock
by Fabio
We collected 1ml of each sample, added 240µl of glycerol (87%) and stored at -80°C.
- BBa_K1033902
- BBa_K1033905
- BBa_K1033910
- BBa_K1033913
- BBa_K1033922
- BBa_K1033925
- BBa_K1033927
- BBa_K1033929
- BBa_K103921
- BBa_J45017
- BBa_K731201
- BBa_K762100
from Liquide Bacterial Cultures transformed the 21st July
3 - Electrophoresis
by Arnaud (Process B), Fabio (gel), Marie and Romain (Process B)
We used 2µL of DNA of the following Biobricks in a 1% Agarose Gel.
Process A
Process B
Results:
- A:
- B:
- C:
4 - Bacterial Culture
by Fabio and Marie
One liquid culture with 5ml of LB (37°C - 150 rpm), IL1403 from the concentrated colony.
5 - Plasmid DNA extraction
by Sean and Terry
Plasmids used: cf part 1
Note: we accidentally used a buffer without 10% ethanol. After removal of the buffer, we repeated the step with a correct version. This may have affected the electrophoresis which followed.
C - Lemon scent
PCR Targeting
by Romain
Strains used: DY330. Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)
Step 1 - Dilution of 300µl of bacterial culture in 30ml of LB + 30µl Cm at 30°C.
Step 2 - With the pre-culture:
- make glycerol stock: 1ml bacterial culture with 240µl glycerol 87%.
- Make extraction of 5ml of plasmid Protocol
Step 3 - When the culture OD650 = 0,6 (Step 1):
- put in ice during 10min.
- centriguge 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 30ml glycerol 10% COLD.
- centriguge 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% COLD.
- centriguge 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% COLD.
Step 4 - Transformation:
- control 50µl (without DNA)
- 50µl transformed bacteria + 2,5µl plasmid + 7,5µl pOSV 230 PCR (purifié)
Step 5 - Electroporation control
Members there:
- Instructors and advisors: Solenne.
- Students: Arnaud, Fabio, Juliette, Mathieu, Marie, Pierre, Romain, Sean and Terry.
