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Nucleic Acid Blotting

After electrophoresis, nucleic acids are transferred from the gel to a solid support (membrane) where they are immobilized. Neutral or positively-charged nylon are the most common types of membrane used for nucleic acid transfer.

Either before or during the blotting step, DNA larger than 15 kb may be fragmented by acid treatment, which depurinates the DNA, breaking it into smaller pieces for more efficient transfer from the gel. DNA may also be denatured to single strands in the gel to improve binding of the negatively charged DNA to a positively charged membrane, and also improve hybridization to the probe. Alkaline denaturation also destroys any residual RNA that may still be present in the DNA.

The conventional method of blotting is capillary transfer, although vacuum blotting is growing in popularity due to the fast and even nature of the transfer.

Vacuum Blotting Capillary Blotting

The agarose gel is placed on a membrane supported by a porous screen. A vacuum is applied under the support screen, drawing the nucleic fragments out of the gel and onto the membrane. Vacuum blotting simplifies treatment of the gel with solutions used in depurination and denaturing steps.

After blotting the membrane is baked in a vacuum or regular oven, or exposed to ultraviolet radiation to permanently attach the transferred DNA or RNA to the membrane.

The agarose gel is mounted onto a paper wick that dips into a reservoir containing transfer buffer. The membrane is sandwiched between the gel and a stack of absorbent material that serves to draw the transfer buffer through the gel by capillary action. The nucleic acid molecules are carried through the gel by the buffer and become immobilized onto the membrane.

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