Standard hybridization technique

Standard hybridization technique

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.