Search Legislation

Commission Directive 2006/56/ECShow full title

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

 Help about what version

What Version

  • Latest available (Revised)
  • Original (As adopted by EU)
 Help about advanced features

Advanced Features

 Help about opening options

Opening OptionsExpand opening options

 Help about UK-EU Regulation

Legislation originating from the EU

When the UK left the EU, legislation.gov.uk published EU legislation that had been published by the EU up to IP completion day (31 December 2020 11.00 p.m.). On legislation.gov.uk, these items of legislation are kept up-to-date with any amendments made by the UK since then.

Changes over time for: Appendix 7

 Help about opening options

Status:

EU Directives are published on this site to aid cross referencing from UK legislation. Since IP completion day (31 December 2020 11.00 p.m.) no amendments have been applied to this version.

Appendix 7Validated reagents for FISH test

1.Oligo-probesU.K.

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 solutionU.K.

[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 HybmixU.K.

NaCl2,7 M
Tris-HCl60 mM (pH 7,4)
EDTA (filter sterilised and autoclaved)15 mM

Dilute to 1x as required.

4.Hybridisation solutionU.K.

1x Hybmix

Sodium dodecyl sulphate (SDS)0,01 %
probe EUB 3385 ng/μl
probe CMSCY3015 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 slides8 slides
Sterile UPW50,1200,4
3x hybmix30,0120,0
1 % SDS0,93,6
Probe EUB 338 (100 ng/μl)4,518,0
Probe CMSCY301 (100 ng/μl)4,518,0
Total volume (μl)90,0360,0

NB. Store all solutions containing light sensitive oligo-probes in the dark at -20 °C. Protect from direct sunlight or electric light during use.U.K.

5.0,1M Phosphate buffer, pH 7,0U.K.

Na2HPO48,52 g
KH2PO45,44 g
Distilled water1,00 L

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

Back to top

Options/Help