3-4.2.1 Procedure:

1. The high-molecular-weight DNA strands are fractioned using restriction enzymes.

2. The DNA fragments are separated based on size by agarose gel electrophoresis.

3. The gel with the restricted fragments is then laid on a filter paper wick which serves as a connection between the membrane and the high salt buffer.

4. The nitrocellulose membrane is placed on top of the gel and a tower of filter papers is used to cover it and these are kept in place with a weight. The capillary action drives the buffer soaking through the filter paper wick, through the gel and the membrane and into the paper towels. Along with the buffer passing through the gel the DNA fragments are also carried with it into the membrane and they bind to the membrane. This causes an effective transfer of fragments (up to 15 kb in length taking around 18hours or overnight.

For DNA fragments larger than 15 kb, before blotting an acid such as diluted HCl is used to treat the gel that depurinates the DNA fragments causing breakage of DNA into smaller pieces, resulting in more efficient transfer from the gel to membrane.

Now a days blotting is also done by applying electric field. This electro blotting technique depends upon current and transfer buffer solution to nucleic acids onto a membrane. Following electrophoresis, a standard tank or semi-dry blotting transfer system is set up. A stack is put together in the following order from cathode to anode: sponge, three sheets of filter paper soaked in transfer buffer gel, PVDF or nitrocellulose membrane, three sheets of filter paper soaked in transfer buffer and then again sponge. Importantly the membrane should be located between the gel and the positively-charged anode, as the current and sample will be moving in that direction. Once the stack is prepared, it is placed in the transfer system, and suitable current is applied for a specific period of time according to the materials being used.