Team:Paris/August 19

From 2008.igem.org

Revision as of 15:04, 19 August 2008 by Hs00590 (Talk | contribs)

← Yesterday

↓ Calendar ↑

Tomorrow →


Ligation FC

Screening of the cloning of E0240 and FlhDC+promotor

Strain Resistance Ligation DNA cloned vector size of the fragment amplified by VF & VR
S159.1 kanamycine L139.1 E0240 (GFP tripart) pSB3K3 1192 bp
S161.1 ampicilline L142.7 FlhDC+promotor pSB1A2 about 1,5 kb

The PCR screening of the transformants L139 and L142 of august 15th revealed several bands for a given clone including one band appearing at the right size.
There are 2 hypothesis:

  • The right clone was contaminated by a wrong one
  • The clone contains 2 plasmids: one with the insert and another one without the insert

In order to check these 2 hypothesis and to isolate (if it is possible) the right clone (containing the plasmid with the insert). We decided to spread the "clone" in question in a LB plate in order to carry out a PCR screening on single colonies.

  • Take of some bacteria from the glycerol stock
  • Resuspension in 400 microL of LB+antibiotic
  • Spreading of 200 microL in a LB agar plate containing the appropriate antibiotic
  • Incubation overnight at 37°C