Team:Paris/August 19

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*Minipreps of L133.1 and L134.2 will be sequenced.
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*==>Minipreps of L133.1 and L134.2 will be sequenced.
=Screening of the cloning of E0240 and FlhDC+promotor=
=Screening of the cloning of E0240 and FlhDC+promotor=

Revision as of 20:01, 4 September 2008

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Contents

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

Electrophoresis

Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13
sample 1 kb
DNA ladder 		control +
pSB3K3
(S158) 		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

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.
Miniprep Glycerol Stock Ligation Name
MP155 S154 L133.1 OmpR*
MP156 S155 L134.2 EnvZ*
  • ==>Minipreps of L133.1 and L134.2 will be sequenced.

Screening of the cloning of E0240 and FlhDC+promotor

Spreading the clones in order to obtain single colonies

Strain Resistance Ligation DNA cloned vector expected size of the fragment amplified by VF & VR mesured size
S159.1 kanamycine L139.1 E0240 (GFP tripart) pSB3K3 1192 bp 1,5 kb
1,1 kb
0,6 kb
S161.1 ampicilline L142.7 FlhDC+promotor pSB1A2 1403 bp 1,4
0,4 kb
0,3 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


Promoter characterization plasmids

Ligation

Protocol


Ligation name Vector digestion Vector description Vector volume Insert digestion Insert description Insert volume Product description Antibiotic
L155 D164 J23101 promoter 10 D163 gfp generator 2 J23101 promoter-gfp generator Amp
L156 D161 pTet promoter 1 D163 gfp generator 4 pTet promoter-gfp generator Kana
D161 1 Vector autoligation control Kana
L157 D125.2 B0015 3 D162 4 tetR tetR-B0015 Amp
D125.2 3 Vector autoligation control Amp

Transformation

Protocol

These transformations were made during the day at 16°C

Digestion

Measurement of concentration of minipreps

to be modified standard protocol

Plasmid Miniprep Concentration (µg/mL) ratio 260/280
MP3 3 38 1.72
MP3 4 30 1.70
MP101 1 333 1.68
MP101 2 416 1.66
MP101 4 200 1.74
MP104 1 145 1.63
MP104 3 147 1.29
MP104 4 51 1.66
MP114 1 173 1.75
MP114 2 263 1.43
MP119 1 42 1.62
MP119 2 26 1.56
MP119 3 39 1.64
MP143 1 138 1.69
MP143 2 150 1.61
MP163 1 80 1.61
MP163 2 79 1.69

Digestion

to be modified

Protocol Digestion

Plasmid Description Miniprep used Enzymes
MP3.3 ? B0015 (double terminator B0010-B0012) - FV 4 EcoRI and XbaI
MP119 promoter J23101 - BV 1 SpeI and PstI
MP104 PTet (Tet promoter) - BV 1 SpeI and PstI
MP114 TetR - FI 1 EcoRI and SpeI
MP143 gfp generator - BI 2 SpeI and PstI

We had a problem with a gel and we lost these digestions.

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