User Manual

7 - DNA EXTRACTION PROTOCOL

We strongly recommend that you read the complete protocol before

beginning. Follow the suggested protocol scrupulously. The volume of the

sample of wine varies according to the intention of the operator and the

information expected.

• 1.8 mL for fermenting grape must, for heavy wine during the

wine-making or maturing process.

• 45 mL for slightly heavy wine, close to bottling or already bottled, the

analysis of which requires a high level of analytic sensitivity.

Preparing the trial

Preparing the equipment

• Switch on the heating block and set it to 95°C. Check that the

temperature has stabilised before continuing with the DNA extraction.

• Prepare the number of purification columns necessary (= Number of

samples to be analysed) by placing each column in a recuperation vial.

Preparing the reagents

• Dilute the W1 washing solution to 1/100 in a sufficient quantity for the

samples to be analysed. See the Tables to prepare the required quantity

of W1 washing solution at 1X dilution on the basis of the number of

samples. (Appendix 1: 1.8 mL volume of analysed grape must/wine and

Appendix 2: 45 mL volume of analysed wine).

• Homogenise buffer R1: put the vial on a magnetic stirring apparatus. During

pipetting, make sure that R1 is continuously being magnetically stirred so

that the lysis reagent is quite homogenous. Use a cone with a wide enough

opening (i.e. a 200 μL to 1000 μL pipette with the corresponding cone).

1°) - Concentration and washing of micro-organism cells from wine

Protocol “1.8 mL for fermenting grape must or for heavy wine during the

wine-making or maturing process”

1. Cell concentration

• Take a 1.8 mL sample of grape must/wine to be analysed and put it in a

2 mL tube with a sterile screw top

• Centrifuge: 5 minutes, at 6000 x g, at 4°C

• Dispose of the supernatant fluid