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Team:Paris Saclay/Notebook/July/30
From 2014.igem.org
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Revision as of 14:00, 4 August 2014
Contents |
Wednesday 30th July
Lab Work
A - The frame coli Odor free
Preparation of electrocompetent cells
by Romain
Strain used: E. coli MG1655Z1 and E. coli MG1655.
Protocol:
Two dilution of 500µl 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 Romain
Strain used: E. coli MG1655Z1 and E. coli MG1655. Plasmid used: BT340 (code for a flipase for E. coli)
Make 2 electroporations in cold electroporation cuvettes:
- A control cuvette(without DNA): 50µl of E. coli MG1655Z1 or E. coli MG1655.
- A second cuvette: 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 cuvette and transfer in 2 tubes.
Incubate during 1h at 30°C.
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 - Salicylate Inducible Suppressing System
DNA purification gel agarose
by Fabio
Once the segregate process made yesterday by electrophoresis, the DNA correspondent to the core of BBa_J61051 was purified from the gel agarose.
Ligation
by Fabio
The final step to have a BioBrick Assembly is the Ligation reaction.
TODO: illustration of the process
- BioBrick BBa_J61051 (Salicylate promoter + NahR) as Part A
- BioBrick BBa_K228001 (RNA suppressor) as Part B
BioBrick Assembly - Ligation Protocol
D - Lemon scent
PCR of BBa_K762100
by Sean
This was a second attempt at yesterday's PCR, with several changes in parameters. Two tubes were prepared: one with yesterday's enzyme and another with a newer version of the same enzyme in order to determine if yesterday's enzyme was one of the causes of the rather unsatisfactory results.
Oligonucleotides used: iPS66, iPS67
Oligonucleotides were diluted twice from 100µM to 50µM.
Protocol
Add into each PCR tube the following:
| Component | For a total volume of 50μl |
|---|---|
| H2O | 35.5μl |
| Phusion buffer 5X | 10μl |
| dNTPs | 1μl |
| iPS66 | 1μl |
| iPS67 | 1μl |
| BBa_K762100 | 1μl |
| Phusion DNA polymerase | 0.25μl |
Tube was placed in PCR machine with the following parameters.
| Cycle step | Temperature | Time | Cycle |
|---|---|---|---|
|
Initial denaturation |
98°C |
1 min |
1 |
| Denaturation | 98°C | 15 s | 25 - 30 |
| Annealing | 52°C | 25 s | 25 - 30 |
| Extension | 72°C | 45 s | 25-30 |
| Final extension | 72°C | 10 min | 1 |
| Final extension | 8°C | hold | 1 |
Members there:
- Instructors and advisors: Alice, Solenne and Sylvie.
- Students: Arnaud, Fabio, Romain, Sean and Terry.
