Dandekar & Chandler:AHL Extraction: Difference between revisions

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== AHL Signal measuring  ==


Assay for acyl-HSL detection using E. coli reporters containing pQF50- (QS-controlled lacZ fusion, Ampicillin) and pJN105- (arabinose inducible R gene, gentamicin) derived plasmids.
== AHL Signal Extraction  ==


===Principle===
In a large test tube containing the sample you wish to test:


 
* Add 5mL of ethyl acetate and vortex on high for 30seconds
 
* Let the sample sit for 10 minutes to allow the two phases to separate out. 
===Materials Needed===
* After the the 10 minute rest, pipette off the clear acetate layer only. Be careful to not draw up any of the bottom cell debris layer.  
 
* Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap. 
* Spectrophotometer capable of reading 520 nm
* Repeat steps 1-4 and add acetate supernatant to tube from first cycle.  
* Vortex
* Place acetate filled tubes into N2 evaporator on medium until all acetate has evaporated off.
* 10mL test tube
* Suspend dried signal in 100uL of ethyl acetate, screw sealable cap on tightly, wrap in parafilm and place in -20 freezer.
* Sterile Cuvets
* 5ml glass pipet
* Nanopure H20
* Chloroform
 
===Protocol===
# Inoculate overnight culture of reporter in LB + Ap 100 Gen 20 at 37C.
# Subculture to desired volume at low density (~0.05) incubate at 37C with shaking
# At OD600 ~0.2-0.3 induce R expression by adding 0.25% arabinose, continue shaking at 37C
# At OD600 ~0.5, aliquot 0.5 ml volumes into eppenforf tubes containing the acyl-HSL sample/standard, shake eppendorf tubes for 2 hours at 37C
# For ease of handling large numbers of eppendorf tubes, we load them in a rack, wrap saran wrap around the tubes + rack, secure the ends with packing tape, and shake the entire rack in the 37C shaker. Saran wrap can be easily cut off using scissors at the end of the experiment.  
# After 2 hours of growth in the presence of acyl-HSL, prepare samples for β-gal assay.  
We lyse cells with 10% vol (50 µl) of chloroform followed by 5 sec vortex and 5 min incubation at RT. 10 µl are then assayed for β-gal using the Tropix kit/luminescence linked detection (10 µl sample, add 70 µl of substrate dilute 1:100 in diluent buffer, incubate 1 hour RT in dark, add 100 µl of accelerator, read luminescence in Tecan plate reader).

Revision as of 16:40, 14 November 2013

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AHL Signal Extraction

In a large test tube containing the sample you wish to test:

  • Add 5mL of ethyl acetate and vortex on high for 30seconds
  • Let the sample sit for 10 minutes to allow the two phases to separate out.
  • After the the 10 minute rest, pipette off the clear acetate layer only. Be careful to not draw up any of the bottom cell debris layer.
  • Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap.
  • Repeat steps 1-4 and add acetate supernatant to tube from first cycle.
  • Place acetate filled tubes into N2 evaporator on medium until all acetate has evaporated off.
  • Suspend dried signal in 100uL of ethyl acetate, screw sealable cap on tightly, wrap in parafilm and place in -20 freezer.
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