UNIT 2.6 Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
Published Online: 1 AUG 2002
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 AUG 2002
- Published Print: JUL 2002
This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The 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 () 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 (), b-agarase digestion of the gel (first ), or via glass beads extraction (second ). Removing linkers from a fragment using a column rather than a gel is described, followed by a method for estimating DNA concentrations in solution.