Bio-Rad VINEO™ Brettanomytest PCR Kit - Manual (Page 7)

We strongly recommend that you read the complete protocol before
beginning the trial.
Follow the suggested protocol scrupulously.
7 - SAMPLING AND TRANSPORT OF SAMPLES
Wine samples are collected under aseptic conditions in sterile glass or
polyethylene recipients or recipients made out of similar material.
The samples must be submitted to the laboratory as quickly as possible,
preferably within 24 and not more than 48 after taking the sample. If the
samples are transported and analysed within 24 h, transport and storage take
place at ambient temperature (+18°C to +30°C). If the samples are
transported and analysed within 48 h, transport and storage take place at
between +2°C to +8°C.
8 - DNA EXTRACTION
DNA extraction must be carried out using the kit developed by Bio-Rad to
extract DNA from wine sample: VINEO™ Extract DNA Kit (ref. Bio-Rad
354-8100).
9 - REAL-TIME PCR
1. Starting up the PCR apparatus
Switch the thermocyler and the computer on in that order and start up the
Opticon Monitor software. For more information and for the definition of
software parameters, see the user guide for the Bio-Rad thermocycler for the
VINEO™ Brettanomytest PCR Kit.
2. Preparing PCR reactions
2.1 Prepare the VINEO™ Brettanomytest PCR Kit reagent mixture by mixing
(40 μL/sample) the amplification solution (tube with white stopper) and
(5 μL/sample) the fluorescent probes (tube with pink stopper). Carry out
the reagent mixture on the basis of the number of samples and controls
to be analysed (at least 1 duplicate (2) of the positive Qs PCR control and
a negative control must be used per plate). The PCR amplification
reagent mixture preparation table in Appendix A indicates the required
quantities of each reagent to be mixed on the basis of the number of
samples to be analysed. Do not forget to include the 2 control points.
Note: do not mix reagent batches.
7
© 2010 Bio-Rad