# 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, Appendix 3: Buffers for test procedures

Extent: E+W+S+N.I.

GENERAL: Unopened sterilized buffers can be stored for up to one year.
1.
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.
Na2HPO4 (anhydrous)
4,26 g
KH2PO4
2,72 g
Distilled water
1.00 L
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.
Additional components may be useful as follows:
For use with homogenisation extraction method.
Purpose
Quantity (per L)
Lubrol flakes
Deflocculant
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.
Na2HPO4.12H2O
2,7 g
NaH2PO4.2H2O
0,4 g
Distilled water
1,00 L
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.
2.
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.
Na2HPO4.12H2O
2,7 g
NaH2PO4.2H2O
0,4 g
NaCl
8,0 g
Distilled water
1.0 L
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.
Na2HPO4.12H2O
3,2 g
NaH2PO4.2H2O
0,15 g
Glycerol
50 ml
Distilled water
100 ml
Anti-fading mountant solutions are commercially available e.g. Vectashield® (Vector Laboratories) or Citifluor® (Leica).

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Source: https://www.legislation.gov.uk/eudr/2006/56/annex/I/appendix/3 (the official version)
Page: https://openlegislation.uk/eu-directive-2006-56/annex-I/appendix-3
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.