Protocol 1.
Generic ITS-PCR fungi
Generic ITS-PCRs will be performed with DNA Engine (PTC-200) instrument (Veenendaal, Nederland) in 96-wells-plates as described below. In all cases 1 µl of the DNA extract will be used in 24 µl master mix, and all samples will be tested in duplo. Negative controls with Milli-Q water will be used in each run. ITS-PCR will be performed with Roche mastermix (Almere, Nederland). Samples will be analyzed on agarose gel and stained with GelRed. The positive or negative result will be base don presence/absence of the amplicon of correct size. PCR Reaction (25 µl): Mastermix per reaction Roche buffer (10x) dNTP (600 µM) ITS1 (5 µM) ITS4 (5 µM) Taq polymerase (5 U/µl) Water
Mastermix 24 µl and 1 µl target Final conc 2.5 µl 1x 2.5 µl 60 µM 1.0 µl 200 nM 1.0 µl 200 nM 0.125 µl 0.625 U 16.875 µl
Forward Primer ITS1 Reverse Primer ITS4
5’- TCCGTAGGTGAACCTGCGG-3’ 5’- TCCTCCGCTTATTGATATGC-3’
PCR program: 2 min 94°C 35 cycles: 30 sec 30 sec 60 sec 5 min 72°C
94°C 56°C 72°C
Rapid diagnostic tools for Phytophthora on horticultural crops 2010
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