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Council Directive 93/85/EECDangos y teitl llawn

Council Directive 93/85/EEC of 4 October 1993 on the control of potato ring rot

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[F1Appendix 3 Buffers for test procedures

Textual Amendments

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

1. Buffers for extraction procedure U.K.

1.1. Extraction buffer (50 mM phosphate buffer, pH 7,0) U.K.

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

Na 2 HPO 4 (anhydrous) 4,26 g
KH 2 PO 4 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:

a

For use with homogenisation extraction method.

Purpose Quantity (per L)
Lubrol flakes Deflocculant a 0,5 g
DC silicone antifoam Anti-foam agent a 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) U.K.

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

Na 2 HPO 4 .12H 2 O 2,7 g
NaH 2 PO 4 .2H 2 O 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 U.K.

2.1. IF-Buffer (10 mM phosphate buffered saline (PBS), pH 7,2) U.K.

This buffer is used for dilution of antibodies.

Na 2 HPO 4 .12H 2 O 2,7 g
NaH 2 PO 4 .2H 2 O 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 U.K.

This buffer is used to wash slides.

Add 0,1 % Tween 20 to the IF buffer.

2.3. Phosphate buffered glycerol, pH 7,6 U.K.

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

Na 2 HPO 4 .12H 2 O 3,2 g
NaH 2 PO 4 .2H 2 O 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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