Unit

UNIT 10.6A Southern Blotting

  1. Terry Brown

Published Online: 1 MAY 2001

DOI: 10.1002/0471142735.im1006as06

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Brown, T. 2001. Southern Blotting. Current Protocols in Immunology. 6:III:10.6A:10.6.1–10.6.13.

Author Information

  1. University of Manchester Institute of Science and Technology, Manchester, United Kingdom

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JUN 1993

Abstract

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.