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Team:Paris Saclay/Notebook/July/28
From 2014.igem.org
(→E - Salicylate Inducible Suppressing System) |
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==Lab work== | ==Lab work== | ||
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===A - The frame=== | ===A - The frame=== | ||
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Protocol: | Protocol: | ||
| - | Two dilution of 200µl of bacterial culture MG1655Z1 and E. coli MG1655 in | + | Two dilution of 200µl of bacterial culture MG1655Z1 and E. coli MG1655 in 15ml of LB at 30°C for each strain. |
When the culture OD<sub>650</sub> = 0,6: | When the culture OD<sub>650</sub> = 0,6: | ||
*put in ice during 10min. | *put in ice during 10min. | ||
*centriguge at 4°C, 5min, 4000rpm. | *centriguge at 4°C, 5min, 4000rpm. | ||
| - | *Discard the supernatant, and resuspend the pellet in | + | *Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% '''COLD'''. |
*centriguge at 4°C, 5min, 4000rpm. | *centriguge at 4°C, 5min, 4000rpm. | ||
*Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% '''COLD'''. | *Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% '''COLD'''. | ||
| Line 38: | Line 37: | ||
After that, add 1ml of cold LB in each tank and transfer in 2 tubes. | After that, add 1ml of cold LB in each tank and transfer in 2 tubes. | ||
| - | Spread on | + | Spread on 8 dishes LB + Cm: |
*20µl of control E. coli MG1655Z1 (without plasmid) | *20µl of control E. coli MG1655Z1 (without plasmid) | ||
Revision as of 07:28, 29 July 2014
Contents |
Monday 28th July
Lab work
A - The frame
Preparation of electrocompetent cells
by Arnaud & Romain
Strain used: E. coli MG1655Z1 and E. coli MG1655.
Protocol:
Two dilution of 200µl of bacterial culture MG1655Z1 and E. coli MG1655 in 15ml of LB at 30°C for each strain.
When the culture OD650 = 0,6:
- put in ice during 10min.
- centriguge at 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% COLD.
- centriguge at 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 15ml glycerol 10% COLD.
- centriguge at 4°C, 5min, 4000rpm.
- Discard the supernatant, and resuspend the pellet in 200µl glycerol 10% COLD.
Transformation of electrocompetent cells
by Arnaud & Romain
Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT640 (code for a flipase for E. coli)
Make 2 électroporations in cold electroporation tanks:
- A control tank (without DNA): 50µl of E. coli MG1655Z1 or E. coli MG1655.
- A second tank: 50µl of E. coli MG1655Z1 or E. coli MG1655 culture + 1µl of BT640 plasmid.
Electroporation : 2500V, 132W, 40µF.
After that, add 1ml of cold LB in each tank and transfer in 2 tubes.
Spread on 8 dishes LB + Cm:
- 20µl of control E. coli MG1655Z1 (without plasmid)
- 50µl of transformed E. coli MG1655Z1 with BT340.
- 100µl of transformed E. coli MG1655Z1 with BT340.
- Transformed and concentrated E. coli MG1655Z1 with BT340.
- 20µl of control E. coli MG1655 (without plasmid)
- 50µl of transformed E. coli MG1655 with BT340.
- 100µl of transformed E. coli MG1655 with BT340.
- Transformed and concentrated E. coli MG1655Z1 with BT340.
Incubate for a night at 30°C.
C - Lemon scent
Liquid Culture
by Fabio
Due to an widespread infection of the cells made the 25th July, we collected the original strain DY330 to verify it's legitimacy.
2ml LB + 20μl of DY330. We incubate cultures at 30°C.
E - Salicylate Inducible Suppressing System
Plasmid DNA Purification
by Fabio
- BBa_J61051
- BBa_K228001
from Bacterial Culture made the 25th July.
Digestion
by Fabio
Once we have a good amount of our BioBricks' plasmid, it's time to do the digestion.
TODO: Digestion's protocol
Electrophoresis
by Fabio
The Electrophoresis was used to verify the success of the Digestion and the success of the plasmid DNA purification at the same time.
- BBa_J61051 Cl.1
- BBa_J61051 Cl.2
- BBa_K228001 Cl.1
- BBa_K228001 Cl.2
Results:
(TODO: Photos from LU000101 to LU000105)
Members present:
- Instructors and advisors: Solenne and Sylvie.
- Students: Arnaud, Fabio, Romain, Sean and Terry.
