DNA detection

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 µg/ml (Boehringer Mannheim)

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

Fluorochrome solution:10 µl anti-Dig-Rhodamin + 5 µl Fluorescein-Avidin in 1000 µl 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)

Steps:

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


CGH CGH