Team:Paris/August 19

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(Difference between revisions)
(Screening of the cloning of E0240 and FlhDC+promotor)
(Screening of the cloning of E0240 and FlhDC+promotor)
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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.
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
*Take of some bacteria from the glycerol stock
-
*Resuspension in 400 microL of LB+antibiotic
+
*Resuspension in 400 µL of LB+antibiotic
-
*Spreading of 200 microL in a LB agar plate containing the appropriate antibiotic
+
*Spreading of 200 µL in a LB agar plate containing the appropriate antibiotic
*Incubation overnight at 37°C
*Incubation overnight at 37°C

Revision as of 15:06, 19 August 2008

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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 µL of LB+antibiotic
  • Spreading of 200 µL in a LB agar plate containing the appropriate antibiotic
  • Incubation overnight at 37°C