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,500μg/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 Na₂ HPO₄ ,pH 7.2.

Pre-hybridisation and hybridisation of blots is carried out using a Hybaid rotary hybridisation oven and bottles at 60℃ (Northern blots or RNA dot blots)or 65℃ (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℃

3 x 20 minutes in 50ml 0.2 x SSC,0.1% SDS at 65℃

2 x 10 minutes in 50ml 0.1 x SSC at 37℃

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℃.

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