3.3.4. Hybridizing the DIG-labelled DNA Probe to DNA on the Blot

This procedure relies on the Roche Applied Science DIG Easy® Hyb, DIG Wash and Block Buffer Set, (with the fluorescent reporter CSPD®).

  1. Pre-warm an appropriate volume of DIG Easy® Hyb solution® to the hybridization temperature.
  2. Pre-hybridize membrane in a small volume of pre-warmed DIG Easy® Hyb solution (20 ml if in a 200 ml hybridization tube).
  3. While the membrane is pre-hybridizing, denature 10 ml of DIG-labelled DNA by boiling for 5 min.
  4. Rapidly cool on ice.
  5. Add appropriate amount of denatured probe to give you (25 ng/ml) into DIG Easy® Hyb solution.
  6. Incubate with agitation in a hybrid oven at 55-58oC for overnight.
  7. Wash membrane in 25-50 ml Washing Solution-1 (2x SSC, 0.1% SDS) 2X for 5 min at room temperature under constant agitation.
  8. Wash membrane in 25-50 ml Washing Solution-2 (0.1% SSC, 0.1% SDS) 2X for 5 min at 68oC under constant agitation.
  9. Wash membranes briefly (1-5 min) in 25 ml of 1X Washing Buffer provided in DIG Wash kit.
  10. Incubate membranes for 30 min in 1X Blocking Solution diluted in maleic acid buffer (supplied in the kit).
  11. Incubate membrane in Anti-body solution for 30 min.
    To make anti-body solution, add 1 µl anti-body to 20 ml 1x blocking solution.
  12. Wash membrane in 1X Washing buffer 2X for 15 min.
    Make sure membrane is immersed in the Washing buffer.
  13. Prepare 20 ml 1X Detection Buffer.
  14. Equilibrate membrane in 20 ml 1X Detection Buffer for 2-5 min.
  15. Transfer the membrane with DNA side facing up to a Plastic wrap that is at least twice the size of the membrane.
  16. Apply 1 ml of CSPD®, ready-to-use (about 20-25 drops) to the membrane.
  17. Quickly cover the membrane with the plastic wrap.
  18. Incubate for 5 min at RT.
  19. Drain off excess buffer by gently brushing across the top of the membrane covered by plastic wrap with a paper towel.
  20. Tape the membrane into a film cassette.
  21. Close the cassette and incubate at 37°C for 10 min to enhance the luminescent reaction.
  22. Remove the film for development using a standard x-ray film developer.