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