Hybrisation of radio-labeled probe to DNA immobilised on nylon membranes
Sodium Phosphate Buffer: 68.4ml 1M Na 2HPO 4, 31.6ml 1M NaH 2PO 4 (pH 6.8)Pre-hybridisation Buffer: 9ml NaPO 4 buffer, 21ml 10% SDS, 60ml 0.5M EDTA, 0.3g BSA
Low stringency wash: 2xSSC, 0.1% SDS
High stringency wash: 0.2xSSC, 0.1%SDS
Membranes are pre-hybridised at 60- 65°C for two hours in Pyrex tubes containing 25ml pre-hybridisation buffer. This is performed in a hybridisation oven containing a rotating spindle (Bachofer). Radiolabeled denatured probe is then added to the Pyrex tube, and returned to the oven. Hybridisation is performed overnight.
The membranes are rinsed with low stringency buffer, and then washed for 20 minutes with 100 ml low stringency buffer in the oven. is was followed by two high stringency washes of 10 minutes each. Following washing, membranes are sealed in cling film. The strength of the hybridisation signal is assessed using a phosphoimager (Molecular dynamics). For re-use, membranes are stripped of probe by subjecting them to two washes of 10 minutes in boiling 0.1% SDS.