Figure 19.4: Preparation of radioactive probe by random primer method.
E. Hybridization and development of Blot:
1. Place the membrane in a hybridization tube and pre-hybridize in 10-20 ml hybridization solution at 650C for atleast 30mins.
2. Preparation of probe for hybridization: Denature the probe by boiling for 10mins and quench on ice.
3. Add the probe to the hybridization solution and incubate over-night. Use high temperature (~650C) for high stringency or moderate temperature (50-550C) for low stringency conditions.
4. Rinse the membrane thoroughly with 10ml of washing solution 2xSSC containing 0.1% SDS at room temperature.
5. Expose the membrane to an x-ray film with an intensifying screen.