# 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 7: Validated reagents for FISH test

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

1.
Oligo-probes
Cms-specific probe CMS-CY3-01:
5′- ttg cgg ggc gca cat ctc tgc acg -3′
Non-specific eubacterial probe EUB-338-FITC:
5′- gct gcc tcc cgt agg agt -3′
2.
Fixative solution
[WARNING! THE FIXATIVE CONTAINS PARAFORMALDEHYDE WHICH IS TOXIC. WEAR GLOVES AND DO NOT INHALE. IT IS ADVISABLE TO WORK IN A FUME CUPBOARD]
(i) Heat nine ml molecular grade water (e.g. Ultra pure water (UPW)) to about 60 °C and add 0,4 g paraformaldehyde. Paraformaldehyde dissolves after adding five drops of 1N NaOH and stirring with a magnetic stirrer.
(ii) Adjust pH to 7,0 by addition of 1ml of 0,1 M phosphate buffer (PB; pH 7,0) and five drops of 1 N HCl. Check pH with indicator strips and adjust if necessary with HCl or NaOH.
[WARNING! DO NOT USE A PH METER IN SOLUTIONS WITH PARAFORMALEDHYDE]
(iii) Filter the solution through a 0,22 µm membrane filter and keep dust-free at 4 °C until further use.
(iv) Note:
Alternative fixative solution: 96 % ethanol.
3.
3x Hybmix
NaCl
2,7 M
Tris-HCl
60 mM (pH 7,4)
EDTA (filter sterilised and autoclaved)
15 mM
Dilute to 1x as required.
4.
Hybridisation solution
1x Hybmix
Sodium dodecyl sulphate (SDS)
0,01 %
probe EUB 338
5 ng/μl
probe CMSCY301
5 ng/μl
Prepare quantities of hybridisation solution according to the calculations in Table. For each slide (containing two different samples in duplicate) 90 μl hybridisation solution is required.
Table:
Suggested quantities for preparation of hybridisation mix
2 slides
8 slides
Sterile UPW
50,1
200,4
3x hybmix
30,0
120,0
1 % SDS
0,9
3,6
Probe EUB 338 (100 ng/μl)
4,5
18,0
Probe CMSCY301 (100 ng/μl)
4,5
18,0
Total volume (μl)
90,0
360,0
NB. Store all solutions containing light sensitive oligo-probes in the dark at -20 °C. Protect from direct sunlight or electric light during use.
5.
0,1M Phosphate buffer, pH 7,0
Na2HPO4
8,52 g
KH2PO4
5,44 g
Distilled water
1,00 L
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.

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Source: https://www.legislation.gov.uk/eudr/2006/56/annex/I/appendix/7 (the official version)
Page: https://openlegislation.uk/eu-directive-2006-56/annex-I/appendix-7
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.