Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/09/17

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09/17/1013

12PM Pulled RhlI out at 12pm. pH looks between 7 and 8 (paper strip)

OD650 GFP pH
0.726 16194 7-8


Spun 1mL of cells, resuspended in 100ul water, checked GFP: 158.
Split cells and LB into four-2mL tubes
Spun at 12.0g for 3min
Transferred 1mL LB supernatant to new tubes

One-2mL tube for later induction (stored at -20)
Three-15mL conical tubes

Added 1mL ethyl acetate to LB

Vortex for 15 seconds, sit for 2min
Repeat 5 times
Fifth time,let sit for 10 minutes

Transfer 1mL of the top layer to 1.5mL tube Let sit for 15-30 seconds Transfer top layer to new 15mL conical tube Store at -20°C

3PM Pulled Rhl2 out

OD650 GFP in LB GFP in H20 pH
0.759 17964 81097 8-9


Repeated extraction procedure

6PM Pulled Rhl3 out

OD650 GFP in LB GFP in H20 pH
0.689 18554 71065 8-9


Repeated the extraction procedure for Rhl3

Tranformations into DH5αT and BL21

  1. J27 (F2620-RFP)
  2. RhlI 8/31/13
  3. EsaI sender A3
  4. GFP-Lux rec 1