Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/03

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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)