Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/07/23

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07/23/2015

  • pEsa_MRV + EsaR had a lot of colonies; backbone controls had fewer colonies.
  • LasR_MRV + pLas has lots of colonies, but its backbones had many as well.

Colony PCR for pEsa.

Reagent Vol (uL) Master Mix (x20)
F Primer 0.5 10
R Primer 0.5 10
DNA - - - -
GoTaq MM 12.5 250
Water 11.5 230
Total: 25 500
  • F Primer - RM seqver mod receiver LuxR home
  • R Primer - RM seqver regulator R
  • 8 colonies were picked for LasR_MRV + pLas ligation
  • 4 colonies were picked for the backbones (4 for ligation and 4 for no ligation)
File:15.07.23 top gel.tiff
File:15.07.23 bottom gel.tiff

Prepare liquid cultures for LasR to digest verify tomorrow. (picked 6 colonies)

Pick more colonies from Dr. Haynes' pAub plate (picked 4 colonies)

Prepare pLux_MRV, LuxR, for ligation

pLux_MRV digestion -
Reagent Volume (uL) '
Vector 7.8 1ug at 127.42ng/uL
10x Buffer 3
EcoRI 1
XbaI 1
Phosphatase 1
Water 16.2
Total 30
LuxR digestion (already PCR purified)
Reagent Volume (uL) '
DNA 5.9 200ng at 34ng/uL
10x Buffer 3
EcoRI 1
XbaI 1
Water 19.1
Total 30
  • Both: Incubate at 37°C for 15 minutes, then heat deactivate.
  • Heat deactivation for E + X is - 80°C for 5 minutes, 65°C for 20 minutes