Pipette 220 µl of lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8,0], 1 mM EDTA [pH 8,0]) into a 1,5 ml Eppendorf tube.

Add 100 µl sample extract and place in a heating block or waterbath at 95 °C for 10 minutes.

Put tube on ice for five minutes.

Add 80 µl Lysozyme stock solution (50 mg lysozyme per ml in 10 mM Tris HCl, pH 8,0) and incubate at 37 °C for 30 minutes.

Add 220 µl of Easy DNA ^® solution A (Invitrogen), mix well by vortexing and incubate at 65 °C for 30 minutes.

Add 100 µl of Easy DNA ^® solution B (Invitrogen), vortex vigorously until the precipitate runs freely in the tube and the sample is uniformly viscous.

Add 500 µl of chloroform and vortex until the viscosity decreases and the mixture is homogeneous.

Centrifuge at 15 000 g for 20 minutes at 4 °C to separate phases and form the interphase.

Transfer the upper phase into a fresh Eppendorf tube.

10.

Add 1 ml of 100 % ethanol ( -20 °C) vortex briefly and incubate on ice for 10 minutes.

11.

Centrifuge at 15 000 g for 20 minutes at 4 °C and remove ethanol from pellet.

12.

Add 500 µl 80 % ethanol ( -20 °C) and mix by inverting the tube.

13.

Centrifuge at 15 000 g for 10 minutes at 4 °C, save the pellet and remove ethanol.

14.

Allow the pellet to dry in air or in a DNA speed vac.

15.

Resuspend the pellet in 100 µl sterile UPW and leave at room temperature for at least 20 minutes.

16.

Store at -20 °C until required for PCR.

17.

Spin down any white precipitate by centrifugation and use 5 µl of the supernatant containing DNA for the PCR.]

[F1ANNEX I U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF THE RING ROT BACTERIUM, CLAVIBACTER MICHIGANENSIS (Smith) Davis et al. ssp. SEPEDONICUS (Spieckermann et Kotthoff) Davis et al. SCOPE OF THE TEST SCHEME

6. PCR TEST U.K.

6.1. DNA purification methods U.K.

6.1.(a) Method according to Pastrik (2000) U.K.