Team:Paris/August 19

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(→Screening of the cloning of E0240 and FlhDC+promotor)
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=Screening of the cloning of E0240 and FlhDC+promotor=
=Screening of the cloning of E0240 and FlhDC+promotor=
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{|border="1" style="text-align: center"
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|'''Strain'''
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|'''Resistance'''
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|'''Ligation'''
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|'''DNA cloned'''
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|'''vector'''
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|'''size of the fragment amplified by VF & VR'''
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|-
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|S159.1
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|kanamycine
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|L139.1
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|E0240 (GFP tripart)
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|pSB3K3
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|1192 bp
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|-
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|S161.1
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|ampicilline
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|L142.7
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|FlhDC+promotor
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|pSB1A2
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|about 1,5 kb
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|}
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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.
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<br>There are 2 hypothesis:
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*The right clone was contaminated by a wrong one
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*The clone contains 2 plasmids: one with the insert and another one without the insert
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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.
 +
*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

Revision as of 15:04, 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 microL of LB+antibiotic
  • Spreading of 200 microL in a LB agar plate containing the appropriate antibiotic
  • Incubation overnight at 37°C
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