Why do you use dbm instead of ncf in the northern blotting and what are the pre-

Why do you use dbm instead of ncf in the northern blotting and what are the pre-treatments required for this paper to be used?

Question Posted / priyanka wadhwa

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Why do you use dbm instead of ncf in the northern blotting and what are the pre-treatments required ..

Answer / Kuldeep Malik

In Northern Blotting, DNA is being transferred from a gel to a membrane. DIG (DNA labeling and detection kit) or DNP (Dinitrophenyl) labeled probes are often used as they allow for enhanced sensitivity and ease of visualization compared to radioactive labels.

DBM (dinitobenzocyclohexanoic anhydride) is a chemical reagent used to label the DNA probe with DNP groups, making it visible under UV light after blotting. On the other hand, NCF (nitrocellulose filter paper) is not commonly used in Northern Blotting as it is more brittle than nylon membranes and may break during the transfer process.

Pre-treatments for the paper include:

1. Soaking the membrane in methanol to fix the RNA onto the membrane.
2. Crosslinking the RNA onto the membrane using UV light or baking the membrane at high temperature.
3. Pre-hybridization to block any unspecific binding sites.

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