Isolation and purification of large DNA restriction fragments from agarose gels.

ABSTRACT

This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first basic 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 is also described and provides relatively high yields for fragments smaller than 2000 bp. Protocols are also provided for separating fragments larger than 1000 bp using low gelling/melting agarose gels and purified by phenol extraction, b-agarase digestion of the gel, or via glass beads extraction. Sieving agarose gels can also be used to resolve very small DNA fragments. Removing linkers from a fragment using a column rather than a gel is included, followed by a method for estimating DNA concentrations in solution.