"
Team:Paris Saclay/Notebook/July/25
From 2014.igem.org
(→Preparation and Transformation of competent cells) |
m (→Friday 25th July) |
||
| (12 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
| + | {{Team:Paris_Saclay/notebook_header}} | ||
=Friday 25th July= | =Friday 25th July= | ||
| - | |||
==Lab work== | ==Lab work== | ||
| + | ===The frame coli Odor free=== | ||
| + | ====PCR on odor-free E. coli ==== | ||
| + | ''by Sean'' | ||
| + | |||
| + | We want to be sure that the odor-free E. coli really ''is'' E. coli. We will use a postitive control. | ||
| + | |||
| + | We have the expected result on electrophoresis | ||
| + | |||
| + | ===Salicylate Inducible Suppressing System=== | ||
| + | ====Bacterial culture:==== | ||
| + | ''by Fabio'' | ||
| + | |||
| + | To insure a resonate quantity of DNA, we made two liquid cultures from each box of the [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/24#Transformation_of_competent_E.coli_cells last process], resulting in a total of 8 liquid cultures composed by 5ml of LB, 5µl of Amp, and respective colonies (4 with '''BBa_J61051''' and 4 with '''BBa_K228001''') (at 9am - 37°C - 150 rpm). | ||
| + | |||
| + | The cultures number 1 and 3 were collect from concentrated colonies' boxes and number 2 and 4, from undiluted colonies' boxes. At the end of the process, tubes 1 and 3 resulted in BioBricks' clones 1 and tubes 2 and 4, BioBricks' clones 2. | ||
| - | === | + | ===Lemon scent=== |
====Results PCR targeting ==== | ====Results PCR targeting ==== | ||
| Line 15: | Line 30: | ||
====Preparation of electrocompetent cells==== | ====Preparation of electrocompetent cells==== | ||
| - | + | ''by Romain'' | |
Strain used: DY330. | Strain used: DY330. | ||
| Line 37: | Line 52: | ||
Strain used: DY330. Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli) | Strain used: DY330. Plasmid used: pJBEI-6409 (code for enzymes to produce limonene from glucose in E. coli) | ||
| - | Make 2 électroporations in cold electroporation | + | Make 2 électroporations in cold electroporation cuvettes: |
| - | *A control | + | *A control cuvette(without DNA): 50µl of DY330. |
| - | *A second | + | *A second cuvette: 50µl of DY330 culture + 1µl of pJBEI-6409 plasmid. |
Electroporation : 2500V, 132W, 40µF. | Electroporation : 2500V, 132W, 40µF. | ||
| - | After that, add 1ml of cold LB in each | + | 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: | Spread on 4 dishes LB + Cm: | ||
| - | |||
*100µl of control (without plasmid) | *100µl of control (without plasmid) | ||
*50µl of transformed DY330 with pJBEI-6409 | *50µl of transformed DY330 with pJBEI-6409 | ||
| Line 54: | Line 68: | ||
Incubate for the weekend at 30°C. | Incubate for the weekend at 30°C. | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
==Reunion== | ==Reunion== | ||
''With Arnaud, Fabio, Sean, Terry, Alice, Solenne, Sylvie & Philippe'' | ''With Arnaud, Fabio, Sean, Terry, Alice, Solenne, Sylvie & Philippe'' | ||
| + | |||
| + | |||
| + | ==Photo of the Day== | ||
| + | [[File:Paris Saclay 25_july.jpg|600px|center]] | ||
'''Members present''': | '''Members present''': | ||
* Instructors and advisors: Solenne. | * Instructors and advisors: Solenne. | ||
* Students: Arnaud, Fabio, Juliette, Pierre, Romain, Sean and Terry. | * Students: Arnaud, Fabio, Juliette, Pierre, Romain, Sean and Terry. | ||
| - | + | {{Team:Paris_Saclay/notebook_footer}} | |
| - | + | ||
Latest revision as of 15:42, 14 October 2014
Contents |
Friday 25th July
Lab work
The frame coli Odor free
PCR on odor-free E. coli
by Sean
We want to be sure that the odor-free E. coli really is E. coli. We will use a postitive control.
We have the expected result on electrophoresis
Salicylate Inducible Suppressing System
Bacterial culture:
by Fabio
To insure a resonate quantity of DNA, we made two liquid cultures from each box of the last process, resulting in a total of 8 liquid cultures composed by 5ml of LB, 5µl of Amp, and respective colonies (4 with BBa_J61051 and 4 with BBa_K228001) (at 9am - 37°C - 150 rpm).
The cultures number 1 and 3 were collect from concentrated colonies' boxes and number 2 and 4, from undiluted colonies' boxes. At the end of the process, tubes 1 and 3 resulted in BioBricks' clones 1 and tubes 2 and 4, BioBricks' clones 2.
Lemon scent
Results PCR targeting
by Romain
After the night, the electrophoresis revealed the oligonucleotides iPS70 and iPS71 prepared with the GoTaq enzyme.
We made a PCR Clean-up. Protocol
After that, we made another electrophoresis which revealed successfully the purification.
Preparation of electrocompetent cells
by Romain
Strain used: DY330.
Protocol:
Dilution of 300µl of bacterial culture DY330 in 30ml of LB at 30°C.
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 30ml 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: 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.
Reunion
With Arnaud, Fabio, Sean, Terry, Alice, Solenne, Sylvie & Philippe
Photo of the Day
Members present:
- Instructors and advisors: Solenne.
- Students: Arnaud, Fabio, Juliette, Pierre, Romain, Sean and Terry.
