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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 plates obtained from the speading of yesterday can't be used because there are not single colonies.
We have to try again, but with a stronger dilution of the bacteria or with a smaller volume of spreading.
- Resuspension of some bacteria from the glycerol stock into 1 mL of LB+antibiotic
- Dilution 10 and dilution 100
- Spreading of 100 µL of each dilution on a LB plate containing the right antibiotic
- Overnight incubation (37°C)
Miniprep and stock glycerol
Stock Glycerol of New Biobrick
Stock number
| Biobricks
| Description
|
S163.1
| B0032
| RBS
|
S163.2
|
S164.1
| E0422
| RBS+ ECFP+ LVA+ term
|
S164.2
|
S165.1
| E0430
| RBS+ YFP+ LVA- term
|
S165.2
|
S166.1
| E0432
| RBS+ YFP LVA+ term
|
S166.2
|
S167.1
| E0420
| RBS+ ECFP LVA- term
|
S167.2
|
S168.1
| I732078
| RBS+ mRFP LVA+ term
|
S168.2
|
Miniprep of New Biobricks
Miniprep
| Biobricks
| Description
|
MP163.1
| B0032
| RBS
|
MP163.2
|
MP164.1
| E0422
| RBS+ ECFP+ LVA+ term
|
MP164.2
|
MP165.1
| E0430
| RBS+ YFP+ LVA- term
|
MP165.2
|
MP166.1
| E0432
| RBS+ YFP LVA+ term
|
MP166.2
|
MP167.1
| E0420
| RBS+ ECFP LVA- term
|
MP167.2
|
MP168.1
| I732078
| RBS+ mRFP LVA+ term
|
MP168.2
|
Construction of pFlgA - YFP tripart (+/- LVA)
Aim : Construction of "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)
Digestion
Measurement of concentration of minipreps
standard protocol
Miniprep
| Biobrick
| C° (µg/mL)
| ratio 260/280
|
MP164.1
| E0422
| 95
| 1.69
|
MP164.2
| E422
| 90
| 1.76
|
MP165.1
| E0430
| 131
| 1.74
|
MP165.2
| E0430
| nd
| nd
|
MP166.1
| E0432
| 112
| 1.65
|
MP166.2
| E0432
| 79
| 1.6
|
MP167.1
| E0420
| 199
| 1.72
|
MP167.2
| E0420
| 194
| 1.73
|
MP168.1
| I732078
| 111
| 1.65
|
MP168.2
| I732078
| 104
| 1.67
|
MP122.1
| E0840
| 56
| 1.58
|
MP122.2
| E0840
| 98
| 1.63
|
Digestion
Protocol Digestion
Name
| Template DNA
| Description
| Vol MP (µl)
| Vol H2O (µl)
| Enzymes
|
D166
| MP165.1
| RBS+ YFP LVA- term - FV
| 7.63
| 17
| EcoRI and XbaI
|
D167
| MP166.1
| RBS+ YFP LVA+ term - FV
| 8.9
| 15.8
| EcoRI and XbaI
|
D131
| MP122.2
| GFP tripart - I
| 10.2
| 14.5
| XbaI and PstI
|
Protocol
Gel Extraction of D166-D167-D131
Well
| 1
| 2
| 3
| 4
| 5
| 6
| 7
| 8
| 9
| 10
| 11
| 12
|
Sample
| 1kb ladder
| MP165.1
| MP166.1
| MP122.2
| no sample
| D166
| no sample
| D167
| no sample
| D131
| no sample
| 100pb ladder
|
Expected size (pb)
|
| 2 957
| 2 996
| 2 957
|
| 2942
|
| 914
|
| 900
|
Measured size (pb)
|
| 3 000
| 3 000
| 3 000
|
| 2500
|
| 2500
|
| 950
|
|
|
Results of the transformation we did yesterday
Number of colonies
Ligation name
| Description
| Antibio
| Number Colonies observed
| Fluorescence
| Comments
|
Ligations
|
L153
| D123(BV) - D165(BI) J23100 - rbs-LasR-Double terminator
| Amp
| 768
| No
| OK
|
L154
| D103(BV) - D165(FV) J23101 - rbs-LasR - Double terminator
| Amp
| 236
| No
| OK
|
Controls
|
C1
| D123(BV)
| Amp
| 23
| No
| OK
|
C2
| D103(FV)
| Amp
| 144
| No
| OK
|
Positive Control
| pUC19
| Amp
| 2264 (efficiency 4,5.10^8)
| No
| OK
|
PCR Screening
==> Protocol
[[Image:]]
Well
| Sample
| Expected size
| Measured size
|
1
| 153.1
| 1194
| 1200
|
2
| 153.2
|
3
| 153.3
|
4
| L153.4
|
5
| L153.5
|
6
| L153.6
|
7
| L153.7
|
8
| L153.8
|
9
| 1kb ladder
|
|
|
10
| L154.1
| 1194
| 1200
|
11
| L154.2
|
12
| L154.3
|
13
| L154.4
|
14
| L154.5
|
15
| L154.6
|
16
| L154.7
|
17
| L154.8
|
Minipreps
Miniprep Name
| Ligation name
| Antibio
| Biobricks
| Description
|
MP169.1
| L153.1
| Amp
|
| J23100 - rbs-LasR-Double terminator
|
MP169.2
| L153.2
|
MP170.1
| L154.1
|
| J23101 - rbs-LasR - Double terminator
|
MP170.2
| L154.2
|
Promoter characterization plasmids
Ligation
Our ligations from yesterday didn't work. The positive control for transformation worked.
Digestion
Transformations
Transformations from yesterday didn't work, and the positive control worked so our ligation didn't work.
|