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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
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Changes over time for: Appendix 7
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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.
| 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 solutionU.K.
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.U.K.
5.0,1M Phosphate buffer, pH 7,0U.K.
| 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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