Saturday, May 15, 2004

Description
DNA detection CGH

Procedure
Wash slides 3 x 3 min at 37C in FA / 2x SSC (new cuvette).
Wash slides 3 x 2 min at 60C in 0.1x SSC (new cuvette).
Keep slides in 4x SSC / 0.1% Tween20 at 37C (new cuvette) until the next step.
Cover each slide with 125 l 3% BSA solution and a 24x60 mm2 cover slip.
Incubate 15 min in a moist chamber at 37C (= blocking step).
Remove cover glasses and place slides in 4x SSC / 0.1% Tween20 (new cuvette) up to the next step.
Centrifuge fluorochromes 3 min at 14000 rpm at 4C
Prepare fluorochrome solution (see above)
Cover each slide with 125 l of the fluorochrome solution and a 24x60 mm2 cover slip. Incubate 20 min in a moist chamber at 37C.
Remove cover slips and wash slides 3 x 3 min at 45C in 4x SSC / 0.1% Tween20.
Incubate slides 5 min in DAPI solution (room temperature).
Mount the slides using DABCO (use about 35 l DABCO per slide).
Store slides at 4C under dark conditions until microscopy.

Recipes
Reagents:

Formamide (FA) / 2x SSC (1:1)

0.1x SSC

4x SSC / 0.1% Tween20

3% BSA in 4x SSC / 0.1% Tween 20

DAPI (4.6-diamidino-2phenylindole dihydrochloride): stock solution 0.2 mg/ml, dissolve 1:5000 in aqua bidest, keep solution in use in a light protected cuvette

anti-Dig-Rhodamin, 200 g/ml (Boehringer Mannheim)

Fluorescein-Avidin-dcs, 1 mg/ml (Vector Laboratories, catalogue-n A-2011)

Fluorochrome solution:10 l anti-Dig-Rhodamin + 5 l Fluorescein-Avidin in 1000 l 3% BSA/4xSSC/0.1%Tween

DABCO (1,4-diazabicyclo2.2.2octane): 2.3% w/v in Glycerol/Tris, e.g. dissolve 23 mg DABCO in 10 ml Glycerol/Tris pH (9 vol Glycerol plus 1 vol Tris 0.2M, pH8)

24x60 mm2 cover slips (e.g. Menzel)


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