Team:Paris/August 19

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(Difference between revisions)
(→Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor)
(→Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor)
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|positive control: <br>pSB3K3 (strain S158)
|positive control: <br>pSB3K3 (strain S158)
|negative control: <br>no template
|negative control: <br>no template
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|colspan="3"|OmpR*
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|colspan="3"|EnvZ*
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|colspan="3"|FlhDC+promotor
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|1 kb DNA ladder
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|'''ligation/clone'''
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|L133.1
|L133.1
|L133.2
|L133.2
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|L132.2
|L132.2
|L132.3
|L132.3
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|1 kb DNA ladder
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|-
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|'''expected size'''
|'''expected size'''
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Revision as of 16:37, 19 August 2008

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Contents

Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor

Electrophoresis

well n° 1 2 3 4 5 6 7 8 9 10 11 12 13
sample 1 kb DNA ladder positive control:
pSB3K3 (strain S158) 		negative control:
no template 		OmpR* EnvZ* FlhDC+promotor 1 kb DNA ladder
ligation/clone L133.1 L133.2 L133.3 L134.1 L134.2 L134.3 L132.1 L132.2 L132.3 expected size 316 bp 0 kb about 1 kb about 1,5 kb 1210 bp
measured size 1 kb 0 kb 1 kb 0 kb 0 kb 0 kb 1,4 kb <0,5 kb 1 kb 1 kb 1 kb
Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor

The clones L133.2 and L133.3 didn't grow up in LB+ampicillin: they seem not to have the plasmid, as revealed by PCR.

Minipreps and glycerol stock

Screening of the cloning of E0240 and FlhDC+promotor

Spreading the clones in order to obtain single colonies

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
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