Answer Posted / Sanjeev Kumar Sharma

"Several factors influence the conditions and time requirements during Northern Blotting:n
1. Gel Electrophoresis: The time for electrophoresis depends on the size of the RNA molecules, voltage, buffer type, pH, and other factors.n
2. Denaturation: Generally, denaturation is performed at high pH (above 8) for 10-30 minutes, or by heating to 65°C-70°C for about 15 minutes.n
3. Transfer (Blotting): Capillary transfer usually takes several hours, while vacuum blotters can transfer RNA in as little as 20-30 minutes.n
4. Hybridization: The time required for hybridization depends on the temperature, probe concentration, and specific activity of the labeled probe.n
5. Washing and Detection: After hybridization, the membrane is washed multiple times with buffers containing high salt concentrations to remove unbound probe, followed by detection using autoradiography or chemiluminescence."

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