# Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al., Appendix 7: Validated reagents for FISH test

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

1.
Oligo-probes
R. solanacearum -specific probe OLI-1-CY3: 5′-ggc agg tag caa gct acc ccc-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 9 ml molecular grade water (e.g. Ultra pure water (UPW)) to about 60 °C and add 0,4 g paraformaldehyde. Paraformaldehyde dissolves after adding 5 drops of 1N NaOH and stirring with a magnetic stirrer.
(ii) Adjust pH to 7.0 by addition of 1ml of 0,1M phosphate buffer (PB; pH 7,0) and 5 drops of 1N 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.
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 %
Formamide
30 %,
probe EUB 338
5 ng/μl
probe OLI-1 or OLI-2
5 ng/μl
Prepare quantities of hybridisation solution according to the calculations in Table 1. For each slide (containing 2 different samples in duplicate) 90 μl hybridisation solution is required. IMPORTANT: FORMAMIDE IS VERY TOXIC SO WEAR GLOVES AND TAKE NECESSARY SAFETY PRECAUTIONS!
Table 1
Suggested quantities for preparation of hybridisation mix
NB: Store all solutions containing light sensitive oligo-probes in the dark at -20 °C.
Protect from direct sunlight or electric light during use.
Number of slides:
1
4
6
8
10
Sterile UPW
23,1
92,4
138,6
184,8
231,0
3x hybmix
30,0
120,0
180,0
240,0
300,0
1 % SDS
0,9
3,6
5,4
7,2
9,0
Formamide
27,0
108,0
162,0
216,0
270,0
Probe EUB 338 (100 ng/μl)
4,5
18,0
27,0
36,0
45,0
Probe OLI-1 or OLI-2 (100 ng/μl)
4,5
18,0
27,0
36,0
45,0
Total volume (μl)
90,0
360,0
540,0
720,0
900,0
5.
0,1M Phosphate buffer, pH 7,0
Na 2 HPO 4
8,52 g
KH 2 PO 4
5,44 g
Distilled water
1,00 L
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

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Source: https://www.legislation.gov.uk/eudr/1998/57/annex/II/appendix/7 (the official version)
Page: https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-7
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.