Team:Paris/August 19

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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 vol. (µL) Insert digestion Insert description Insert vol. (µL) 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
TL156 D161 1 Vector autoligation control Kana
L157 D125.2 B0015 3 D162 tetR 4 tetR-B0015 Amp
TL157 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

Protocol Digestion

Plasmid Description Miniprep used Enzymes
MP118.1 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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