Council Directive 98/57/ECShow full title

[F1 7.1. Potato extract fixation U.K.

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

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

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

7.1.3. Remove the supernatant and dissolve the pellet in 200 µl of fixative prepared < 24 hours previously. Vortex and incubate for one hour in the refrigerator. U.K.

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

7.1.5. Spot 16 µl of the fixed suspensions onto a clean multitest slide as shown in Fig. 7.1. Applying two different samples per slide, undiluted and use 10 µl to make a 1:100 dilution (in 0,01 M PB). The remaining sample solution (49 µl) can be stored at -20  °C after addition of one volume of 96 % ethanol. In case the FISH assay requires repeating, remove the ethanol by centrifugation and add an equal volume of 0,01 PB (mix by vortexing). U.K.

Fig. 7.1

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

7.1.6. 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 2006/63/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..