TITLE: Ethanol Quantification assay
AUTHORS: Marcos Valenzuela-Ortega

[DESCRIPTION]
Chemoenzymatic method for quantification of ethanol with a spectrophotometer at 500 nm (not UV !)

Based on Lewicka 2014   (DOI: 10.1021/sb500020g )

Adapted for analysis in plate reader of multiple samples at the same time.

[STEPS]
SECTION: Prepare stocks
2. 
 Prepare the following stocks using your tris buffer:

 
       Stock         mg/mL         mM         Mw            Yeast Adh         5                  NAD+         13.3         20         685.41            PMS         6.1         20         306.34            INTV         25.3         50         505.00      



INTV needs to be dissolved in buffer-DMSO (50:50)

Keep reagentson ice 
(no need for ethanol)

Keep the buffer at Room temperature!

SECTION: Prepare culture samples
1. Sample your culture and spin it 16000 rpm, 1 min.

Transfer  Supernatant to new tube and keep on ice if analysed inmediately, or freeze if analysed in a different day.
SECTION: Prepare reaction mastermix
4. Prepare reaction mix for all your samples. 

Calculate the volume to be prepared should be 200 µL * well * 1.1 (so you have)
When counting "wells", consider replicates for samples and standards.

That is the volum of Tris you need, to which you have to add:
Yeast ADH: 5 µL per mL
NAD+: 1µL per mL
PMS: 1 µL per mL
INTV: 20 µL per mL
SECTION: Prepare stocks
3. Prepare your ethanol standard ladder by making different dilutions of your 1M ethanol with tris buffer. The standard could be, for example, 400-200-100-50-25-10-5-0 mM ethanol.
SECTION: Reaction plate and incubation
5. Plan how to load samples in plate, and annotate it.

Consider that the reaction mix should be loaded to the replicates at different moments (so their variability comprises the variability caused by loading the mix at the different times.)
SECTION: Reaction plate and incubation
6. Mix & incubate
SECTION: Analysis and troubleshooting
8. Blank should be done with water, samples measurements will be reliable only in the linear region of the standard.

BMG plate readers do most of the analysis for you, if you tell the software which wells had each blank, standarda and sample. You can introduce this in the software before or after the readings.

You can have a positive control (negative control culture spiked with ethanol).

Sensitivity can be increased using 20 µL of sample in 200 µL of mastermix, or increasing incubation time to 30 minutes.
SECTION: Reaction plate and incubation
7. Measure Abs 500 nm in plate reader at Room temperature 
 

SECTION: Analysis and troubleshooting
9. 
SECTION: Reaction plate and incubation
5.1. Load 200 µL of buffer to the blank wells. (some replicates here are good too). These wells wont receive reaction mix.

SECTION: Reaction plate and incubation
5.3. Load 200 µL of reaction mix to the wells with your multichannel pipette with as little difference of time between sample as possible.
SECTION: Reaction plate and incubation
5.2. Load 10 µL of sample to the well bottom. Be sure all the volume of liquid is in the well and not in your pipette tip.

SECTION: Reaction plate and incubation
6.1. Mix the plate (the plate reader can do this).
SECTION: Reaction plate and incubation
6.2. Incubate the plate for Room temperature for 15 minutes, in the dark.