- Buffer 1: 100 mM Tris-HCl, pH 7.5; 15 mM NaCl
- Buffer 2: 0.5% (w/v) blocking reagent (Roche or Amersham) in buffer 1. This is dissolved by heating the solution to 50-70 °C for at least 1 h. The prepared solution can then be stored at 4 °C for up to 1 month
- Buffer 3: 100 mM Tris-HCl, pH 9.5; 100 mM NaCl; 50 mM MgCl 2
- Antibody AP-mix: For the detection of biotin use l:500 dilution of anti-biotin conjugated to alkaline phosphatase (Roche, Sigma, Amersham) in buffer 1. For the detection of digoxigenin use 1:5000 dilution of anti-digoxigenin conjugated to alkaline phosphatase (Roche) in buffer 1
- Detection reagents (mix the following immediately prior to use), or purchase as pre-mixed and stabilized solution (Life Technologies):
22.5 m l of NBT solution (4-nitroblue tetrazolium chloride, 75 mg/ml in 70% dimethylformamide; available in solution from Life Technologies). Store frozen in aliquots.
- 17.5 m l of BCIP (5-bromo-4-chloro-2-indolyl-phosphate, 50 mg/ml in 70% dimethylformamide available in solution from Life Technologies). Store frozen in aliquots.
4.96 ml of buffer 3
- Charged nylon membrane as used for Southern hybridization of DNA (e.g. Hybond N+ from Amersham; off-cut pieces are usually suitable).
- Cut the membrane to the size required (e.g. 20 x 20 mm)
- Soak the membrane in buffer 1 for 5 min and then blot dry between filter paper
- Micro-pipette small spots of labeled DNA, and controls of previously tested DNA, solution onto the membrane (0.5-1 m l) and leave to adsorb and partly dry for 5-10 min. Write spot identifications on membrane with pencil
- Place the membrane in a Petri dish with 4 ml buffer 1 for 1 minute, and then place in 4 ml buffer 2 for 30 min. Shake gently
- Pour off buffer and distribute 0.5 ml antibody-AP mixture over the membrane (Note 2), cover with a plastic sheet and incubate at 37 °C for 30 min, shaking gently
- Wash the membrane in buffer 1, 15 min
- Transfer the membrane to buffer 3 for 2 min
- Prepare the detection reagents, pour over membrane and leave for 5-10 min in the dark for the color to develop fully
- Wash the membrane in water and air-dry
- Metal forceps will damage the membrane and cause areas of stain precipitate. Even with plastic forceps, handle gently by corners only
- Increase amount if a larger membrane is used
- Labeled probe is detected by purple-brown staining of the probe spots. Store test membranes in the dark (e.g. inside a notebook), and mark or describe the signal dots since they will fade over time.