{
  "url": "https://openlegislation.uk/eu-directive-2006-56/annex-I/appendix-7",
  "json_url": "https://openlegislation.uk/eu-directive-2006-56/annex-I/appendix-7.json",
  "legislation": "eudr/2006/56",
  "legislation_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",
  "legislation_url": "https://openlegislation.uk/eu-directive-2006-56",
  "path": "annex/I/appendix/7",
  "kind": "appendix",
  "number": "7",
  "heading": "Validated reagents for FISH test",
  "text": "1.\nOligo-probes\nCms-specific probe CMS-CY3-01:\n5′- ttg cgg ggc gca cat ctc tgc acg -3′\nNon-specific eubacterial probe EUB-338-FITC:\n5′- gct gcc tcc cgt agg agt -3′\n2.\nFixative solution\n[WARNING! THE FIXATIVE CONTAINS PARAFORMALDEHYDE WHICH IS TOXIC. WEAR GLOVES AND DO NOT INHALE. IT IS ADVISABLE TO WORK IN A FUME CUPBOARD]\n(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.\n(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.\n[WARNING! DO NOT USE A PH METER IN SOLUTIONS WITH PARAFORMALEDHYDE]\n(iii) Filter the solution through a 0,22 µm membrane filter and keep dust-free at 4 °C until further use.\n(iv) Note:\nAlternative fixative solution: 96 % ethanol.\n3.\n3x Hybmix\nNaCl\n2,7 M\nTris-HCl\n60 mM (pH 7,4)\nEDTA (filter sterilised and autoclaved)\n15 mM\nDilute to 1x as required.\n4.\nHybridisation solution\n1x Hybmix\nSodium dodecyl sulphate (SDS)\n0,01 %\nprobe EUB 338\n5 ng/μl\nprobe CMSCY301\n5 ng/μl\nPrepare 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.\nTable:\nSuggested quantities for preparation of hybridisation mix\n2 slides\n8 slides\nSterile UPW\n50,1\n200,4\n3x hybmix\n30,0\n120,0\n1 % SDS\n0,9\n3,6\nProbe EUB 338 (100 ng/μl)\n4,5\n18,0\nProbe CMSCY301 (100 ng/μl)\n4,5\n18,0\nTotal volume (μl)\n90,0\n360,0\nNB. Store all solutions containing light sensitive oligo-probes in the dark at -20 °C. Protect from direct sunlight or electric light during use.\n5.\n0,1M Phosphate buffer, pH 7,0\nNa2HPO4\n8,52 g\nKH2PO4\n5,44 g\nDistilled water\n1,00 L\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/2006/56/annex/I/appendix/7",
    "retrieved_at": "2026-11-01T15:22:35.204179+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}