Team:Paris Saclay/Notebook/July/25

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(Difference between revisions)
(→Friday 25th July)
(→Preparation and Transformation of competent cells)
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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.
-
====Preparation and Transformation of competent cells====
+
====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 OD<sub>650</sub> = 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 tanks:
 +
 
 +
*A control tank (without DNA): 50µl of DY330.
 +
*A second tank: 50µl of DY330 culture + 1µl of pJBEI-6409 plasmid.
 +
 
 +
Electroporation : 2500V, 132W, 40µF.
 +
 
 +
After that, add 1ml of cold LB in each tank 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.
===E - Salicylate Inducible Suppressing System===
===E - Salicylate Inducible Suppressing System===

Revision as of 20:59, 27 July 2014

Contents

Friday 25th July

Lab work

C - 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 tanks:

  • A control tank (without DNA): 50µl of DY330.
  • A second tank: 50µl of DY330 culture + 1µl of pJBEI-6409 plasmid.

Electroporation : 2500V, 132W, 40µF.

After that, add 1ml of cold LB in each tank 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.

E - Salicylate Inducible Suppressing System

Bacterial culture:

by Fabio

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 24th July

Reunion

With Arnaud, Fabio, Sean, Terry, Alice, Solenne, Sylvie & Philippe

Members present:

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

Back to the calendar

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