Dandekar & Chandler:AHL Extraction: Difference between revisions

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== N-Acyl homoserine lactone (AHL) Extraction ==


== AHL Signal Extraction  ==


===Principle===
Use only glass tubes to reduce loss of signal during extraction and storage!


Assay for acyl-HSL detection using E. coli reporters containing pQF50- (QS-controlled lacZ fusion, Ap) and pJN105- (arabinose inducible R gene, Gm) derived plasmids.
In a large test tube (18 mm) containing the sample you wish to test:


===Materials Needed===
# 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.   
* Spectrophotometer capable of reading 520 nm
# After the 10 minute rest, pipette off the clear acetate layer only being careful to not draw up any of the bottom cell debris layer.  
* Vortex
# Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap. [https://www.fishersci.com/shop/products/fisherbrand-disposable-borosilicate-glass-tubes-polypropylene-screw-cap-6/p-196656 This type of vial] works well. (Fisher 14-962-26F)
* 10mL test tube
# Repeat steps 1-4 and add acetate supernatant to collection tube from step 4.  
* Sterile Cuvets
# Place acetate filled tubes into N2 evaporator until all acetate has evaporated off. (keep on low level to prevent splashing)
* 5ml glass pipet
# Suspend dried signal in 100uL of ethyl acetate, screw sealable cap on tightly, wrap in parafilm and place in -20 freezer.
* Nanopure H20
* 0.2m HCl
* 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 12:21, 11 August 2015

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AHL Signal Extraction

Use only glass tubes to reduce loss of signal during extraction and storage!

In a large test tube (18 mm) containing the sample you wish to test:

  1. Add 5mL of ethyl acetate and vortex on high for 30seconds
  2. Let the sample sit for 10 minutes to allow the two phases to separate out.
  3. After the 10 minute rest, pipette off the clear acetate layer only being careful to not draw up any of the bottom cell debris layer.
  4. Deposit the clear acetate layer in a 10mL glass tube with a sealable air tight cap. This type of vial works well. (Fisher 14-962-26F)
  5. Repeat steps 1-4 and add acetate supernatant to collection tube from step 4.
  6. Place acetate filled tubes into N2 evaporator until all acetate has evaporated off. (keep on low level to prevent splashing)
  7. 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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