From 2008.igem.org
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Revision as of 14:03, 21 August 2008
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Construction of pFlgA - YFP tripart (+/- LVA)
Aim : Construction of "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)
Digestion
Protocol
[[Image:KR00019b.jpg| thumb|Gel Extraction of D166-D167]
Well
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
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Sample
| 1kb ladder
| MP165.1
| MP166.1
| no sample
| D166
| no sample
| D167
| no sample
| 100pb ladder
| no sample
|
Expected size (pb)
|
| 2 957
| 2 996
|
| 2942
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| 2981
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Measured size (pb)
|
| 3 000
| 3 000
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Screening of the cloning of E0240 and FlhDC+promotor
We obtained single colonies with the dilution 100.
13 clones were analysed by PCR
PCR screening
reaction mixture (25 µL)
- 12,5 µL Quick load PCR Mixture 2X
- 0,5 µL O18
- 0,5 µL O19
- 11,5 µL water
PCR screening programm
- elongation time: 1 min 30
- primers: O18 and O19
Gel n°1 (E0240 in pSB3K3)
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well n°
| 1
| 2
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| 8
| 9
| 10
| 11
| 12
| 13
| 14
| 15
| 16
| 17
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sample
| 1 kb DNA ladder
| positive control
| negative control
| S159.1
| 100 bp DNA ladder
|
colonie n°
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| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
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expected size
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measured size
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Gel n°2 (FlhDC+promotor in pSB1A2)
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well n°
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
| 14
| 15
| 16
| 17
|
sample
| 1 kb DNA ladder
| positive control
| negative control
| S159.1
| 100 bp DNA ladder
|
colonie n°
|
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|
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
|
|
expected size
|
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measured size
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