Team:Paris Saclay/Notebook/July/25

From 2014.igem.org

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{{Team:Paris_Saclay/notebook_header}}
=Friday 25th July=
=Friday 25th July=
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==Lab work==
==Lab work==
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===The frame coli Odor free===
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====PCR on odor-free E. coli ====
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''by Sean''
 +
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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
 +
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===Salicylate Inducible Suppressing System===
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====Bacterial culture:====
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''by Fabio''
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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).
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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.
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===C - Lemon scent===
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===Lemon scent===
====Results PCR targeting ====
====Results PCR targeting ====
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After that, we made another electrophoresis which revealed successfully the purification.
After that, we made another electrophoresis which revealed successfully the purification.
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====Preparation and Transformation of competent cells====
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====Preparation of electrocompetent cells====
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''by Romain''
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===E - Salicylate Inducible Suppressing System===
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Strain used: DY330.
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====Bacterial culture:====
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''by Fabio''
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8 liquid cultures with 5ml of LB and 5µl of Amp, 4 with '''BBa_J61051''' and 4 with '''BBa_K228001''' (at 11am - 37°C - 150 rpm), all from the concentrated colony made the [https://2014.igem.org/Team:Paris_Saclay/Notebook/July/24 24th July]
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Protocol:
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Dilution of 300µl of bacterial culture DY330 in 30ml of LB at 30°C.
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 +
When the culture OD<sub>650</sub> = 0,6:
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*put in ice during 10min.
 +
*centriguge at 4°C, 5min, 4000rpm.
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*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====
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''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:
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*100µl of control (without plasmid)
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*50µl of transformed DY330 with pJBEI-6409
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*100µl of transformed DY330 with pJBEI-6409
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*The rest of transformed DY330 with pJBEI-6409 (concentrate in approximately 150µl)
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 +
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''
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==Photo of the Day==
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[[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.
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{{Team:Paris_Saclay/notebook_footer}}
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[https://2014.igem.org/Team:Paris_Saclay/Notebook Back to the calendar]
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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

Paris Saclay 25 july.jpg

Members present:

  • Instructors and advisors: Solenne.
  • Students: Arnaud, Fabio, Juliette, Pierre, Romain, Sean and Terry.

Back to the calendar

Retrieved from "http://2014.igem.org/Team:Paris_Saclay/Notebook/July/25"