Paris/July 17

From 2007.igem.org

(Difference between revisions)
(PCRs)
(Transduction of MG1655 with P1 stock made on w121)
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OD=0.6
OD=0.6
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<br>
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* Control (900µL LB MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
+
* Preparation of samples:
-
* 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
+
** Control (900µL LB MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
-
* 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
+
** 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
-
* 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
+
** 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
-
<br>
+
** 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
-
20mn @ 37°C = adsorption
+
* 20mn @ 37°C = adsorption
* Centrifugation 1mn @ 10000rpm
* Centrifugation 1mn @ 10000rpm
* Resuspension in LB + Na Citrate 20mM + DAP 300µM
* Resuspension in LB + Na Citrate 20mM + DAP 300µM
* 1H growth @ 37°C
* 1H growth @ 37°C
* Plate on LB + Na Citrate + Erythromycin + DAP
* Plate on LB + Na Citrate + Erythromycin + DAP

Revision as of 15:24, 17 July 2007

PCRs

  • The assembly PCRs did not work... we'll try them again !
  • The Lox66-DapAColi PCRs worked very well in all conditions

=> Gel purification

PCR : FtsZ-E.Coli
Name Ftsz-E.Coli
Annealing T° 55°C
Time of elongation 2m30'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 1 Lox71-FtsZ-F 10µM 2.5µL
oligoR 2 FtsZ-R 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA toothpick in glycerol stock of MG1655
PCR : DapA-Subtilis
Name DapA-Subtilis
Annealing T° 55°C
Time of elongation 2m30'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 8 Lox66-DapASubtilis-F 10µM 2.5µL
oligoR 9 DapASubtilis R 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA 0.5µL of Subtilis DNA from stock (DB tube in fridge)
PCR : Assembly PCR DGAT
Name assembly PCR of DGAT
Annealing T° 55°C
Time of elongation 2m30'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 10µM 2.5µL
oligoR 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA 1µL of PCR product DGAT 1 1µL of PCR product DGAT 2
PCR : Assembly PCR FtsZ-FtsA
Name assembly PCR of FtsZ-FstA
Annealing T° 55°C
Time of elongation 2m30'
Number of Cycles 30
Buffer 5x 10µL
MgCl2 10µM 0µL
dNTP 10µM 1µL
oligoF 3 10µM 2.5µL
oligoR 2 10µM 2.5µL
water 34µL
polymerase Phusion 0.5µL
DNA 1µL of PCR product FtsZ 1 1µL of PCR product FtsZ 2

I make a gel to check PCR products. To put on gel :

  • 50µL of PCR product
  • 5µL of "bleu de charge" jaune.

DAP solution contamination test

We spread on LB plates DAP solution :

  • 50µL of H20 (control)
  • 50µL of aliquot DAP 50mM (in use)
  • 50µL of DAP 1mM
  • 50µL of DAP 0.2 mM
  • 50µL of DAP 50mM

+ filtration of the DAP stock

Transduction of MG1655 with P1 stock made on w121


As it still didn't work, we try for now with culture of MG1655 in exponential growth rate (ExpGR)
OD=0.6

  • Preparation of samples:
    • Control (900µL LB MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
    • 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
    • 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
    • 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
  • 20mn @ 37°C = adsorption
  • Centrifugation 1mn @ 10000rpm
  • Resuspension in LB + Na Citrate 20mM + DAP 300µM
  • 1H growth @ 37°C
  • Plate on LB + Na Citrate + Erythromycin + DAP