You will need:
6 x SSC (0.9M NaCl, 90mM sodium citrate, pH 7)
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.
Dr. Simon Dawson,
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Clifton Boulevard ,
Tel: +44 (0)115 9249924 ex.44787,
FAX: +44 (0)115 9422225,
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