Appendix 3 Buffers for test proceduresF1

United Kingdom

GENERAL: Unopened sterilized buffers can be stored for up to one year.

Buffers for extraction procedure

1.1.

Extraction buffer (50 mM phosphate buffer, pH 7,0)

This buffer is used for extraction of the bacterium from plant tissues by homogenisation or shaking.

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.

Additional components may be useful as follows:

	Purpose	Quantity (per L)
For use with homogenisation extraction method.
Lubrol flakes	Deflocculant ^f1	0,5 g
DC silicone antifoam	Anti-foam agent	1,0 ml
Tetrasodium pyrophosphate	Anti-oxidant	1,0 g
Polyvinylpyrrolidone- 40 000 (PVP-40)	Binding of PCR inhibitors	50 g

1.2.

Pellet buffer (10 mM phosphate buffer, pH 7,2)

This buffer is used for resuspension and dilution of potato tuber heel-end core extracts following concentration to a pellet by centrifugation.

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.

Buffers for the IF test

2.1.

IF-Buffer (10 mM phosphate buffered saline (PBS), pH 7,2)

This buffer is used for dilution of antibodies.

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.

2.2.

IF-buffer-Tween

This buffer is used to wash slides.

Add 0,1 % Tween 20 to the IF buffer.

2.3.

Phosphate buffered glycerol, pH 7,6

This buffer is used as a mountant fluid on the windows of IF slides to enhance fluorescence.

Anti-fading mountant solutions are commercially available e.g. Vectashield ^® (Vector Laboratories) or Citifluor ^® (Leica).

Annotations

F1: Substituted by Commission Directive 2006/56/EC of 12 June 2006 amending the Annexes to Council Directive 93/85/EEC on the control of potato ring rot.