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Team:Paris Saclay/Notebook/July/29
From 2014.igem.org
(→Electrophoresis) |
(→PCR of BBa_K762100) |
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===D - Lemon scent=== | ===D - Lemon scent=== | ||
| + | |||
| + | ====Transformation of electrocompetent cells==== | ||
| + | ''by Arnaud & Terry'' | ||
| + | |||
| + | Strain used: DY330. Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli) | ||
| + | |||
| + | Make 2 électroporations in cold electroporation cuvettes: | ||
| + | |||
| + | *A control cuvette(without DNA): 50µl of DY330. | ||
| + | *A second cuvette: 50µl of DY330 culture + 1µl of pJBEI-6409 plasmid. | ||
| + | |||
| + | Electroporation : 2500V, 132W, 40µF. | ||
| + | |||
| + | After that, add 1ml of cold LB in each cuvette and transfer in 2 tubes to incubate during 1 to 2h at 30°C. | ||
| + | |||
| + | Spread on 4 dishes LB + Cm: | ||
| + | |||
| + | *100µl of control (without plasmid) | ||
| + | *50µl of transformed DY330 with pJBEI-6409 | ||
| + | *100µl of transformed DY330 with pJBEI-6409 | ||
| + | *The rest of transformed DY330 with pJBEI-6409 (concentrate in approximately 150µl) | ||
| + | |||
| + | Incubate for the weekend at 30°C. | ||
====PCR of BBa_K762100==== | ====PCR of BBa_K762100==== | ||
Revision as of 08:45, 1 August 2014
Contents |
Tuesday 29th July
Lab Work
A - The frame coli Odor free
PCR
by Romain
Strains used: E. coli MG1655Z1 and E. coli MG1655, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.
Protocol
Add into a PCR tube the following:
| Component | For a total volume of 50μl |
|---|---|
| H2O | 27.25μl |
| Green GoTaq buffer 5X | 10μl |
| dNTPs | 1μl |
| FTR-Apra-F | 2μl |
| FTR-Apra-R | 2μl |
| DMSO | 1.5μl |
| MgCl2 | 4μl |
| Bacterial culture | 2μl |
| Green GoTaq enzyme | 0.25μl |
Tube was placed in PCR machine with the following parameters.
| Cycle step | Temperature | Time | Cycle |
|---|---|---|---|
|
Initial denaturation |
95°C |
5 min |
1 |
| Denaturation | 95°C | 30 s | 30 |
| Annealing | 60°C | 30 s | 30 |
| Extension | 72°C | 2 min | 30 |
| Final extension | 72°C | 5 min | 1 |
| Final extension | 12°C | hold | 1 |
The different bacterial cultures in each tubes:
- Tube 1: MG1655Z1 10I
- Tube 2: MG1655 100II
- Tube 3: MG1655 50I
- Tube 4: MG1655 100I
- Tube 5: MG1655Z1 10II
- Tube 6: MG1655 50II
- Tube 7: MG1655Z1 10I positive control with plasmid
Results of the PCR: The electrophoresis revealed nothing
New PCR with different enyme.
Strains used: MG1655 and MG1655Z1, and oligonucleotides used: FTR-Apra-F and FTR-Apra-R.
Protocol
Add into a PCR tube the following:
| Component | For a total volume of 50μl |
|---|---|
| H2O | 37.25μl |
| DreamTaq buffer 10X | 5μl |
| dNTPs | 1μl |
| FTR-Apra-F | 1μl |
| FTR-Apra-R | 1μl |
| DMSO | 2.5μl |
| Bacterial culture | 2μl |
| DreamTaq enzyme | 0.25μl |
Tube was placed in PCR machine with the following parameters.
| Cycle step | Temperature | Time | Cycle |
|---|---|---|---|
|
Initial denaturation |
95°C |
5 min |
1 |
| Denaturation | 95°C | 30 s | 30 |
| Annealing | 60°C | 30 s | 30 |
| Extension | 72°C | 2 min | 30 |
| Final extension | 72°C | 5 min | 1 |
| Final extension | 12°C | hold | 1 |
- Tube 1: MG1655Z1 10I
- Tube 2: MG1655 100II
- Tube 3: MG1655 50I
- Tube 4: MG1655 100I
- Tube 5: MG1655Z1 10II
- Tube 6: MG1655 50II
- Tube 7: MG1655Z1 10I positive control with plasmid
Results of the PCR: The electrophoresis revealed successfully the DNA!
C - Salicylate Inducible Suppressing System
Digestion
by Fabio
After the experiments done the 28th July, we are sure to manipulate the right plasmid from both BioBricks and also that we have a good concentration of them. Now it's time to start the [http://parts.igem.org/Help:Assembly/3A_Assembly Assembly procedure] in order to concatenate both BioBricks.
- BioBrick BBa_J61051 (Salicylate promoter + NahR)
- BioBrick BBa_K228001 (RNA suppressor)
Electrophoresis
by Fabio
Results:
First process to verify the digestion PHOTO I: LU000107
we are sure that we have the right BioBricks fragments and in a good concentration
Second one to separate both parts (only with J61051). PHOTO II:
D - Lemon scent
Transformation of electrocompetent cells
by Arnaud & Terry
Strain used: DY330. Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli)
Make 2 électroporations in cold electroporation cuvettes:
- A control cuvette(without DNA): 50µl of DY330.
- A second cuvette: 50µl of DY330 culture + 1µl of pJBEI-6409 plasmid.
Electroporation : 2500V, 132W, 40µF.
After that, add 1ml of cold LB in each cuvette and transfer in 2 tubes to incubate during 1 to 2h at 30°C.
Spread on 4 dishes LB + Cm:
- 100µl of control (without plasmid)
- 50µl of transformed DY330 with pJBEI-6409
- 100µl of transformed DY330 with pJBEI-6409
- The rest of transformed DY330 with pJBEI-6409 (concentrate in approximately 150µl)
Incubate for the weekend at 30°C.
PCR of BBa_K762100
by Sean
Oligonucleotides used: iPS66, iPS67
Oligonucleotides were diluted twice from 100µM to 50µM.
Protocol
Add into a 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 | 55°C | variable | 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.
