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Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor
Electrophoresis
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| sample
| 1 kb DNA ladder
| positive control:
pSB3K3 (strain S158)
| negative control:
no template
| OmpR*
| EnvZ*
| FlhDC+promotor
| 1 kb DNA ladder
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| ligation/clone
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| L133.1
| L133.2
| L133.3
| L134.1
| L134.2
| L134.3
| L132.1
| L132.2
| L132.3
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| expected size
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| 316 bp
| 0 kb
| about 1 kb
| about 1,5 kb
| 1210 bp
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| 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
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 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.
| Miniprep
| Glycerol Stock
| Ligation
| Name
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| MP155
| S154
| L133.1
| OmpR*
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| MP156
| S155
| L134.2
| EnvZ*
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- 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
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| S159.1
| kanamycine
| L139.1
| E0240 (GFP tripart)
| pSB3K3
| 1192 bp
| 1,5 kb
1,1 kb
0,6 kb
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| S161.1
| ampicilline
| L142.7
| FlhDC+promotor
| pSB1A2
| 1403 bp
| 1,4
0,4 kb
0,3 kb
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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.
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
| Ligation name
| Vector digestion
| Vector description
| Vector volume
| Insert digestion
| Insert description
| Insert volume
| Product description
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| L155
| D164
| J23101 promoter
| 10
| D163
| gfp generator
| 2
| J23101 promoter-gfp generator
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| L156
| D161
| pTet promoter
| 1
| D163
| gfp generator
| 4
| pTet promoter-gfp generator
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| D161
| 1
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| Vector autoligation control
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| L157
| D125.2
| 3
| D162
| 4
| tetR-B0015
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| D125.2
| 3
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| Vector autoligation control
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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.
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