Team:Paris/August 19

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[[Image:KR000188.jpg|thumb|Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor]]
[[Image:KR000188.jpg|thumb|Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor]]
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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.
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==Minipreps and glycerol stock==
==Minipreps and glycerol stock==
 +
 +
*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 stocks were made for the clones L133.1 and L134.2.
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 +
{|border="1" style="text-align: center"
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|'''Miniprep'''
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|'''Glycerol Stock
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|'''Ligation'''
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|'''Name'''
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|-
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|MP155
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|S154
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|L133.1
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|OmpR*
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|-
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|MP156
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|S155
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|L134.2
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|EnvZ*
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|-
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*Minipreps of L133.1 and L134.2
=Screening of the cloning of E0240 and FlhDC+promotor=
=Screening of the cloning of E0240 and FlhDC+promotor=

Revision as of 17:35, 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

Minipreps and glycerol stock

  • 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 stocks were made for the clones L133.1 and L134.2.
  • Minipreps of L133.1 and L134.2

Screening of the cloning of E0240 and FlhDC+promotor

Spreading the clones in order to obtain single colonies

Miniprep Glycerol Stock Ligation Name
MP155 S154 L133.1 OmpR*
MP156 S155 L134.2 EnvZ*
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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