Dandekar & Chandler:AHL Signal Measuring: Difference between revisions
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# Calculate OD<sub>600</sub> of overnight reporter strain + antibiotics | # Calculate OD<sub>600</sub> of overnight reporter strain + antibiotics | ||
# Subculture to desired volume at low density (~0. | # Subculture to desired volume at low density (~0.025) into 10mL LB + 10 μL Amp100, 10μL Gen 10 incubate at 37°C with shaking | ||
# Add 10μL of ethyl acetate concentrated signal to a small glass vial and allow to evaporate in a well ventilated area | # Add 10μL of ethyl acetate concentrated signal to a small glass vial and allow to evaporate in a well ventilated area | ||
# At OD<sub>600</sub> ~0.2-0.3 induce R expression by adding 0.25% arabinose (25μL in 10mL), continue shaking at 37°C | # At OD<sub>600</sub> ~0.2-0.3 induce R expression by adding 0.25% arabinose (25μL in 10mL), continue shaking at 37°C | ||
Line 49: | Line 49: | ||
# At OD600 ~0.5, aliquot 100μL into the vial containing (now) dried signal, and appropriate volumes for tubes containing NOT containing the acyl-HSL sample/standard | # At OD600 ~0.5, aliquot 100μL into the vial containing (now) dried signal, and appropriate volumes for tubes containing NOT containing the acyl-HSL sample/standard | ||
# Perform desired dilutions across the samples | # Perform desired dilutions across the samples | ||
# Shake eppendorf tubes for | # Shake eppendorf tubes for 1.5 hours at 37°C | ||
# After 2 hours of growth add 10% (10μL in 100μL) chloroform to stop growth and lyse cells) | # After 2 hours of growth add 10% (10μL in 100μL) chloroform to stop growth and lyse cells) | ||
# Shake samples for 30 seconds and let them sit for 10 minutes at room temp. | # Shake samples for 30 seconds and let them sit for 10 minutes at room temp. | ||
# Take the top most | # Take the top most 5μL from each sample and add it to a corresponding well of an Optiplate™ 96 well plate | ||
# Make a 1:100 mixture of Tropix® Galacton-Plus and Tropix® Galacto Reaction Buffer Diluent and add | # Make a 1:100 mixture of Tropix® Galacton-Plus and Tropix® Galacto Reaction Buffer Diluent and add 35μL to each well containing a sample. | ||
# Place the plate in a dark place at room temp for one hour (a seldom opened drawer works well) | # Place the plate in a dark place at room temp for one hour (a seldom opened drawer works well) | ||
# After one hour dark sit, add | # After one hour dark sit, add 50μL of Tropix Accelerator and read on plate reader. (luminescence, 1000ms, 3 second shake between reads) | ||
====C4·HSL via pECP61.5==== | ====C4·HSL via pECP61.5==== |
Revision as of 15:23, 29 April 2015
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.
Principle
(add something here)
Materials Needed
- Spectrophotometer capable of reading 600 nm
- Plate reader
- Vortex
- 10mL test tube(s)
- eppendorf tubes (or chloroform resistant 96well plate)
- Sterile Cuvets
- Glass vial
- Nanopure H20
- Chloroform
- β-gal assay reagents
Protocol
- Inoculate overnight culture of reporter in LB plus respective antibiotics (10 μL Amp, 10μL 100 Gen 20 for pSC11 pJ105L / 10μL Amp 100 for pECP61.5) at 37C.
3OC12·HSL via pSC11 pJ105L
- Calculate OD600 of overnight reporter strain + antibiotics
- Subculture to desired volume at low density (~0.025) into 10mL LB + 10 μL Amp100, 10μL Gen 10 incubate at 37°C with shaking
- Add 10μL of ethyl acetate concentrated signal to a small glass vial and allow to evaporate in a well ventilated area
- At OD600 ~0.2-0.3 induce R expression by adding 0.25% arabinose (25μL in 10mL), continue shaking at 37°C
- During this shaking period label chloroform-resistant tubes or 96 well plate with sample names and dilutions 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 37°C shaker. Saran wrap can be easily cut off using scissors at the end of the experiment.
- At OD600 ~0.5, aliquot 100μL into the vial containing (now) dried signal, and appropriate volumes for tubes containing NOT containing the acyl-HSL sample/standard
- Perform desired dilutions across the samples
- Shake eppendorf tubes for 1.5 hours at 37°C
- After 2 hours of growth add 10% (10μL in 100μL) chloroform to stop growth and lyse cells)
- Shake samples for 30 seconds and let them sit for 10 minutes at room temp.
- Take the top most 5μL from each sample and add it to a corresponding well of an Optiplate™ 96 well plate
- Make a 1:100 mixture of Tropix® Galacton-Plus and Tropix® Galacto Reaction Buffer Diluent and add 35μL to each well containing a sample.
- Place the plate in a dark place at room temp for one hour (a seldom opened drawer works well)
- After one hour dark sit, add 50μL of Tropix Accelerator and read on plate reader. (luminescence, 1000ms, 3 second shake between reads)
C4·HSL via pECP61.5
- Add 10μL of ethyl acetate concentrated signal to a small glass vial and allow to evaporate in a well ventilated area
- Label chloroform-resistant tubes or 96 well plate with sample names and dilutions For ease of handling large numbers of eppendorf tubes, we load them in a rack, wrap saran wrap around the tubes + rack
- Calculate OD600 of overnight reporter strain + antibiotic
- Subculture to desired volume at low density (~0.01) into 10mL LB + 10 μL Amp + 10μL IPTG
- Add 100μL into the vial containing (now) dried signal, and appropriate volumes for tubes containing NOT containing the acyl-HSL sample/standard
- Perform desired dilutions across the samples
- Secure the ends with packing tape, and shake the entire rack in the 37°C shaker. Saran wrap can be easily cut off using scissors at the end of the experiment.
- After 5 hours of growth add 10% (10μL in 100μL) chloroform to stop growth and lyse cells)
- Shake samples for 30 seconds and let them sit for 10 minutes at room temp.
- Take the top most 10μL from each sample and add it to a corresponding well of an Optiplate™ 96 well plate
- Make a 1:100 mixture of Tropix® Galacton-Plus and Tropix® Galacto Reaction Buffer Diluent and add 70μL to each well containing a sample.
- Place the plate in a dark place at room temp for one hour (a seldom opened drawer works well)
- After one hour dark sit, add 100μL of Tropix Accelerator and read on plate reader. (luminescence, 1000ms, 3 second shake between reads)