Council Directive 93/85/EECDangos y teitl llawn

[F1 5.1. Potato extract fixation U.K.

The following protocol is based upon Wullings et al. , (1998): U.K.

5.1.1. Prepare fixative solution (see Appendix 7). U.K.

5.1.2. Pipette 100 µl of each sample extract into an Eppendorf tube and centrifuge for eight min at 7 000  g. U.K.

5.1.3. Remove the supernatant and dissolve the pellet in 500 µl of fixative prepared < 24 hours previously. Vortex and incubate overnight at 4 °C. U.K.

An alternative fixative is 96 % ethanol. To use this dissolve the pellet from step 5.1.2 in 50 µl 0,01M PB and 50 µl 96 % ethanol. Vortex mix and incubate at 4 °C for 30 to 60 minutes.

5.1.4. Centrifuge for 8 min. at 7 000  g, remove the supernatant and resuspend the pellet in 75 µl 0,01M PB (see Appendix 3). U.K.

5.1.5. Spot 16 µl of the fixed suspensions onto a clean multitest slide as shown in Fig. 3. Apply two different samples per slide, undiluted and use 10 µl to make a 1:100 dilution (in 0,01M PB). The remaining sample solution (49 µl) can be stored at -20  °C after addition of 1 volume of 96 % ethanol. In case the FISH assay requires repeating, remove the ethanol by centrifugation and add an equal volume of 0,01M PB (mix by vortexing). U.K.

Figure 3.

Layout for FISH slide

Sample 1	Blank	Blank	Blank	Sample 2
window 1	window 2	window 3	window 4	window 5
Sample 1	Blank	Blank	Blank	Sample 2
window 6	window 7	window 8	window 9	window 10
Coverslip 1			Coverslip 2

5.1.6. Air-dry the slides (or on slide dryer at 37 °C) and fix them by flaming. U.K.

At this stage the procedure may be interrupted and the hybridisation continued the following day. Slides should be stored dust-free and dry at room temperature.]

Textual Amendments

F1 Substituted by 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.