{
  "url": "https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-7",
  "json_url": "https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-7.json",
  "legislation": "eudr/1998/57",
  "legislation_title": "Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.",
  "legislation_url": "https://openlegislation.uk/eu-directive-1998-57",
  "path": "annex/II/appendix/7",
  "kind": "appendix",
  "number": "7",
  "heading": "Validated reagents for FISH test",
  "text": "1.\nOligo-probes\nR. solanacearum -specific probe OLI-1-CY3: 5′-ggc agg tag caa gct acc ccc-3′\nNon-specific eubacterial probe EUB-338-FITC: 5′-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 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.\n(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.)\n(iii) Filter the solution through a 0,22 µm membrane filter and keep dust-free at 4 °C until further use.\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 %\nFormamide\n30 %,\nprobe EUB 338\n5 ng/μl\nprobe OLI-1 or OLI-2\n5 ng/μl\nPrepare 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!\nTable 1\nSuggested quantities for preparation of hybridisation mix\nNB: Store all solutions containing light sensitive oligo-probes in the dark at -20 °C.\nProtect from direct sunlight or electric light during use.\nNumber of slides:\n1\n4\n6\n8\n10\nSterile UPW\n23,1\n92,4\n138,6\n184,8\n231,0\n3x hybmix\n30,0\n120,0\n180,0\n240,0\n300,0\n1 % SDS\n0,9\n3,6\n5,4\n7,2\n9,0\nFormamide\n27,0\n108,0\n162,0\n216,0\n270,0\nProbe EUB 338 (100 ng/μl)\n4,5\n18,0\n27,0\n36,0\n45,0\nProbe OLI-1 or OLI-2 (100 ng/μl)\n4,5\n18,0\n27,0\n36,0\n45,0\nTotal volume (μl)\n90,0\n360,0\n540,0\n720,0\n900,0\n5.\n0,1M Phosphate buffer, pH 7,0\nNa 2 HPO 4\n8,52 g\nKH 2 PO 4\n5,44 g\nDistilled water\n1,00 L\nDissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "F",
      "label": "F1",
      "text": "Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/1998/57/annex/II/appendix/7",
    "retrieved_at": "2026-11-01T15:23:06.931886+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."
}