Council Directive 98/57/ECShow full title

[F1 (a) Method according to Pastrik (2000) U.K.

1)

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.

2)

Add 100 µl sample extract and place in a heating block or water bath at 95 °C for 10 min.

3)

Put tube on ice for 5 min.

4)

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 min.

5)

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

6)

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.

7)

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

8)

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

9)

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 min.

11)

Centrifuge at 15 000  g for 20 min 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 min 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.]

Textual Amendments

F1 Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al..