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Team:Paris Saclay/Notebook/July/30
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=Wednesday 30th July= | =Wednesday 30th July= | ||
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==Lab Work== | ==Lab Work== | ||
| - | + | ===The frame coli Odor free=== | |
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====Preparation of electrocompetent cells==== | ====Preparation of electrocompetent cells==== | ||
''by Romain'' | ''by Romain'' | ||
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Incubate for a night at 30°C. | Incubate for a night at 30°C. | ||
| - | === | + | ===Salicylate Inducible Suppressing System=== |
====DNA purification gel agarose==== | ====DNA purification gel agarose==== | ||
''by Fabio'' | ''by Fabio'' | ||
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[https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Ligation BioBrick Assembly - Ligation Protocol] | [https://2014.igem.org/Team:Paris_Saclay/Protocols/BioBrick_Assembly#Ligation BioBrick Assembly - Ligation Protocol] | ||
| - | === | + | ===Lemon scent=== |
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====PCR of BBa_K762100==== | ====PCR of BBa_K762100==== | ||
''by Sean'' | ''by Sean'' | ||
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| Annealing | | Annealing | ||
| 52°C | | 52°C | ||
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| 25 - 30 | | 25 - 30 | ||
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| + | ==Photo of the Day== | ||
| + | [[File:Paris Saclay 30_july.jpg|600px|center]] | ||
'''Members there''': | '''Members there''': | ||
* Instructors and advisors: Alice, Solenne and Sylvie. | * Instructors and advisors: Alice, Solenne and Sylvie. | ||
* Students: Arnaud, Fabio, Romain, Sean and Terry. | * Students: Arnaud, Fabio, Romain, Sean and Terry. | ||
| - | + | {{Team:Paris_Saclay/notebook_footer}} | |
Latest revision as of 15:44, 14 October 2014
Contents |
Wednesday 30th July
Lab Work
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.
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
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 |
Photo of the Day
Members there:
- Instructors and advisors: Alice, Solenne and Sylvie.
- Students: Arnaud, Fabio, Romain, Sean and Terry.
