From 2008.igem.org
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| + | ==='''Measurement of the concentration of D166 & D167 purified'''=== |
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| + | {|border="1" style="text-align: center" |
| + | |'''Digestion Name''' |
| + | |'''Concentration (µg/mL)''' |
| + | |'''Ratio 260/280''' |
| + | |- |
| + | |D166 |
| + | |11 |
| + | |4.73 |
| + | |- |
| + | |D167 |
| + | |10 |
| + | |2.84 |
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Revision as of 19:33, 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
|
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
|
| 2981
|
|
Measured size (pb)
|
| 3 000
| 3 000
|
|
|
|
|
|
|
|
Measurement of the concentration of D166 & D167 purified
Digestion Name
| Concentration (µg/mL)
| Ratio 260/280
|
D166
| 11
| 4.73
|
D167
| 10
| 2.84
|
Ligation
Protocol
Ligation Name
| Vector Name
| Volume Vector (µL)
| Insert
| Volume Insert (µL)
|
L160
| D166
| 3.63
| D132
| 2.03
|
L161
| D167
| 4.00
| D132
| 2.01
|
Control L160
| D166
| 3.63
| -
| -
|
Control L161
| D167
| 4.00
| -
| -
|
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
- positive control: S158 (pSB3K3)
- negative control: no template
Electrophoresis
Gel n°1 (E0240 screening)
Gel n°2 (FlhDC+promotor screening)
- 1% agarose gel
- 10 µL loaded
Gel n°1 (E0240 in pSB3K3)
|
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°
|
|
|
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
|
|
expected size
|
|
|
| 1192 bp
|
|
measured size
|
|
|
| 1,5 kb 1,1 kb 0,6 kb
|
|
Gel n°2 (FlhDC+promotor in pSB1A2)
|
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
| S161.1
| 100 bp DNA ladder
|
colonie n°
|
|
|
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
| 13
|
|
expected size
|
|
|
| 1403 bp
|
|
measured size
|
|
|
| 0,3 kb
|
|
Results:
- The clone of E0240 (S159.1) always have several bands amplified by PCR. It might contain different plasmids.
- The clone of FlhDC+promotor (S161.1) don't have the correct size band. It also doesn't have the insert in the plasmid.
|