07/3/2015
Ligate modular receiver vector (MRV) + RhlR, LuxR, and EsaR.
1. Digest 1ug MRV
Reagent
|
Vol (uL)
|
DNA |
1.45 (1ug at 691.8ng/uL)
|
EcoRI |
1
|
XbaI |
1
|
Phosphotase |
1
|
FD Buffer |
3
|
Water |
22.55
|
Total: |
30
|
- Incubate at 37°C for 15 minutes.
- Heat inactivate at
- 80°C for 5 minutes
- 65°C for 20 minutes
2. Digest PCR amplified and purified, regulators w/ E+X.
Reagent
|
Vol (uL)
|
DNA |
variable; 200ng
|
EcoRI |
1
|
XbaI |
1
|
FD Buffer |
3
|
Water |
21.3
|
Total: |
30
|
Amount of DNA used for each reaction w/r to their concentrations:
RhlR
|
EsaR
|
LuxR
|
3.7uL |
3.6uL |
5.9uL
|
3. Ligate 3:1 ratio of insert:bb
- Amounts of DNA used were based on vector/insert size.
- Vector is ~3,200bp. Transcription factor inserts are ~851bp.
Reagent
|
Volume (uL)
|
'
|
|
EsaR |
RhlR |
LuxR |
Vector-only
|
Insert DNA (40ng) |
6uL |
6uL |
6uL |
0uL
|
Vector DNA (50ng) |
1.52uL |
1.52uL |
1.52uL |
1.52uL
|
10x Buffer |
2uL |
2uL |
2uL |
2uL
|
Ligase |
1uL |
1uL |
1uL |
1uL
|
Water |
9.48uL |
9.48uL |
9.48uL |
15.48uL
|
Total |
20uL |
20uL |
20uL |
20uL
|
4. Transformation with DH5αT cells
- 10uL of each ligation reaction were used in the transformation.
PCR amplify inducible promoters for:
- Aub, Bta, Cer, and Exp.
- (Bta, Cer, and Exp seemed to be running low; unsure if these will work)
|