Check Lux components
- Single colony PCR for :
- 4 different colonies of S0168 (from a transformation plate from 12/08)
- 4 different colonies of J04630 (from a transformation plate from 12/08)
- Protocol : add 1μL of cells (diluted in water), 10μL of Master Mis, 7μL of SDW, 1μL of VF primer and 1μL of VR primer
- Gel PCR products
Gel 1
- Lane2 : Hyperladder1
- Lane3 : JO4630, colony 1
- Lane4 : JO4630, colony 2
- Lane5 : JO4630, colony 3
- Lane6 : JO4630, colony 4
- Lane7 : HyperladderI
Gel 2
- Lane2 : HyperladderI
- Lane3 : ECE190 double digest
- Lane4 : S0168, colony 1
- Lane5 : S0168, colony 2
- Lane6 : S0168, colony 3
- Lane7 : S0168, colony 4
- Lane8 : HyperladderI
- Lane9 : R0040
- Lane10 : R0062
- Lane11 : Ladder 100bp
- Results
- R0040 and R0062 : one big band of about 300b (expected size 293), OK!
- S0168 : one band of about 400b for the 4 different colonies (expected size 1234!), bad! This plate does not contain S0168
- J04630 (colonies 2 and 4) : one band of about 1100b (expected size 1173), OK!
- J04630 (colony 1) : one good band plus another band...
- J04630 (colony 3) : one band of about 600b, bad!
Ligation
- Materials :
- AgrA
- AgrB
- AgrC
- AgrD
- Pupp
- Pspac
- Ppac
- Pxyl
- RBS S
- RBS W
- psB4C5
- Double digest of PCR products
- Run vector, AgrA and AgrD on a gel
- DNA clean and concentrator for AgrA, B,C and D, promoters
- Microclean for both RBS
- Nanodrop
|
260/280 |
ng/μL
|
AgrA |
1.66 |
16.4
|
AgrB |
1.91 |
23.5
|
AgrC |
1.99 |
35.9
|
AgrD |
2.13 |
4.9
|
Pxyl |
1.54 |
5.6
|
Ppac |
1.49 |
4.6
|
Pspc |
1.62 |
9.6
|
Pupp |
1.88 |
8.5
|
RBS S |
2.44 |
29
|
RBS W |
1.44 |
10.7
|
- Extract plasmid annd Agr from gel and clean
- Ligation
|