Council Directive 98/57/ECShow full title

[F1 7.2. Hybridisation U.K.

7.2.1. Dehydrate the cells in a graded ethanol series of 50 %, 80 % and 96 % for one minute each. Air dry the slides in a slide-holder. U.K.

7.2.2. Prepare a moist incubation chamber by covering the bottom of an air-tight box with tissue or filter paper soaked in 1x hybmix (Appendix 7). Pre-incubate the box in the hybridisation oven at 45 °C for at least 10 minutes. U.K.

7.2.3. Apply 10 μl of hybridisation solution (Appendix 7) to eight windows (windows 1, 2, 4, 5, 6, 7, 9 and 10; see Fig 7.1) of each slide leaving the two centre windows (3 and 8) empty. U.K.

7.2.4. Apply coverslips (24 × 24 mm) to the first and last four windows without trapping air. Place the slides in the pre-warmed moist chamber and hybridise for five hours in the oven at 45 °C in the dark. U.K.

7.2.5. Prepare three beakers containing 1 l of Milli Q (molecular grade) water, 1 l of 1x hybmix (334 ml 3x hybmix and 666 ml Milli Q water) and 1 l of 1/8x hybmix (42 ml 3x hybmix and 958 ml Milli Q water). Pre-incubate each in a waterbath at 45 °C. U.K.

7.2.6. Remove the coverslips from the slides and place the slides in a slide holder. U.K.

7.2.7. Wash away excess probe by incubation for 15 minutes in the beaker with 1x hybmix at 45 °C. U.K.

7.2.8. Transfer the slide holder to 1/8 hybmix washing solution and incubate for a further 15 minutes. U.K.

7.2.9. Dip the slides briefly in Milli Q water and place them on filter paper. Remove excess moisture by covering the surface gently with filter paper. Pipette 5 to 10 μl of anti-fading mountant solution (e.g. Vectashield, Vecta Laboratories, CA, USA or equivalent) on each window and apply a large coverslip (24 × 60 mm) over the whole slide.] U.K.

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..