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Standard hybridization technique

Standard hybridization technique

The standard solution typically used for both pre-hybridisation and hybridisation is based on that given in Maniatis et al., (1982) with both Denhart's solution and heterologous DNA being replaced by heparin (Singh and Jones, 1984).

You will need:

6 x SSC (0.9M NaCl, 90mM sodium citrate, pH 7)
10% SDS
50mg/ml heparin sulphate (Sigma)

Standard hybridisation solution: 6 x SSC, 500ug/ml heparin, 1% SDS.

N.B: A modification used for the hybridisation buffer which can also give extremely good results is a variation on that described by Church and Gilbert, 1984. The buffer contains 7% SDS and 0.25 M Na2HPO4, pH 7.2.

Pre-hybridisation and hybridisation of blots is carried out using a Hybaid rotary hybridisation oven and bottles at 60°C (Northern blots or RNA dot blots) or 65°C (plaque hybridisations or DNA dot blots).

1) Place membrane in the hybridisation bottle with the minimum amount of hybridisation solution (0.1ml/cm2 membrane).

2) Pre-hybridise at the appropriate temperature for at least 3 hours.

3) Boil the radiolabelled probe for 3 minutes to denature the DNA.

N.B: Care should be taken when handling radioactive sources. Appropriate protective clothing and radiation monitoring equipment should be used at all times.

4) Add probe to the hybridisation bottle to give a final concentration of approximately 1-5 x 105 cpm/ml (by Cerenkov counting) of hybridisation fluid. Allow hybridisation to continue for 6 hours or more, typically overnight.

5) Once hybridisation is complete, the hybridisation solution is carefully removed and the membrane washed, sequentially, in the bottle in the following fashion:-

3 x 10 minutes in 50ml 2 x SSC, 0.1% SDS at 65°C
3 x 20 minutes in 50ml 0.2 x SSC, 0.1% SDS at 65°C
2 x 10 minutes in 50ml 0.1 x SSC at 37°C

N.B: Optimum temperatures for each probe-target hybridisation reaction will obviously have to be determined empirically.

6) After washing, remove the membrane and air dry. Wrap the membrane in Saran Wrap and autoradiograph at -80°C.

If the membrane is required for re-probing, the air drying step should be omitted and the membrane autoradiographed wet.

Keywords: hybridization, DNA

Contact Email Address:

Submitted at 17:06 on 22/1/96 by:

Dr. Simon Dawson,
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Q.M.C. ,
Clifton Boulevard ,
NG7 2UH,
Tel: +44 (0)115 9249924 ex.44787,
FAX: +44 (0)115 9422225,

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