From 2008.igem.org
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| | |'''DNA cloned''' | | |'''DNA cloned''' |
| | |'''vector''' | | |'''vector''' |
| - | |'''size of the fragment amplified by VF & VR''' | + | |'''expected size of the fragment amplified by VF & VR''' |
| | |- | | |- |
| | |S159.1 | | |S159.1 |
Revision as of 17:38, 19 August 2008
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Screening of the cloning of OmpR*, EnvZ* and FlhDC+promotor
Electrophoresis
| well n°
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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
|
| ligation/clone
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|
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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
|
| 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.
| 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
|
| S159.1
| kanamycine
| L139.1
| E0240 (GFP tripart)
| pSB3K3
| 1192 bp
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| 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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