Paris/July 18

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Contents

Transduction of w121 strain

Titration of the stock of phage from 12.7.7. : ~109 phages/ml : OK

FtsZTS strain screening

We want to test these strains :

  • 121.4
  • 121.5
  • 121.6
  • 129
  • 1.129
  • DCR 14.1
  • DCR 14.2
  • DCR 14.3

I made glycerol stock of these strains, stored in the freezer. Test :

  • Spread these strains on two plates (preheated before spreading) :
    • One at 30°C
    • One at 42°C

The results tomorrow.

Minipreps

We have 2 clones for each plasmid :

  • E0422
  • E0241
  • E0840
  • B0030
  • J61047

Elution within 50µL H2O
DNA is within the Miniprep box in the -20°C fridge.

Digestion products

Photos !!!

DAP solution contamination test

Results :

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

PCRs

PCR : Assembly PCR Lox71-Fts1-FTsZ1 + FtsZ2
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 2580
50, 55, 60, 65°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 3 Lox71-FtsA-F 2.5µL NO
3m00' Oligo R 10µM 2 FtsZ-R 2.5µL Image (click to enlarge)
Number cycles Water 30µL [[Image:|30px]]
35 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Lox71-FtsA-FtsZ1 2µL + FtsZ2 2µL

E.coli pKS::DGAT

  1. After overnight culture (see July 17), we do not observe significant triglyceride synthesis.
  1. We decide to test in solid culture and wait some days. E.coli transformed by pKS::DGAT and the negative control (E.coli transformed by part B0015) were spread on LB medium +- oleate 2mM +- IPTG (0.4mM).
  1. We try in minimum medium (see Protocols) replacing lactose/galactose by 2mM oleate, too.