Appendix 7 Validated reagents for FISH testF1
United Kingdom
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.