Unit

UNIT 2.6 Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels

  1. David Moore1,
  2. Dennis Dowhan1,
  3. Joanne Chory2,
  4. Randall K. Ribaudo (NA-45 paper)3

Published Online: 1 AUG 2002

DOI: 10.1002/0471142727.mb0206s59

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Moore, D., Dowhan, D., Chory, J. and Ribaudo, R. K. 2002. Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels. Current Protocols in Molecular Biology. 59:II:2.6:2.6.1–2.6.12.

Author Information

  1. 1

    Baylor College of Medicine, Houston, Taxas

  2. 2

    The Salk Institute, La Jolla, California

  3. 3

    National Institute of Allergy and Infectious Diseases, Bethesda, Maryland

Publication History

  1. Published Online: 1 AUG 2002
  2. Published Print: JUL 2002

Abstract

This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first protocol describes electroelution of the fragment of interest from standard agarose gels using buffer-filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA-45 paper (second protocol) provides relatively high yields for fragments <2000 bp. Fragments >1000 bp can also be separated on low gelling/melting agarose gels and purified by phenol extraction (third protocol), b-agarase digestion of the gel (first alternate protocol), or via glass beads extraction (second alternate protocol). Removing linkers from a fragment using a column rather than a gel is described, followed by a method for estimating DNA concentrations in solution.