Separation of Reaction Products on a Sequencing Gel

Abstract

A mixture of millions of different fragments of genomic DNA produced by the restriction-endonuclease digest and the chemical sequencing reactions must be separated by size on polyacrylamide gels. Therefore a very high standard is required when preparing and running the sequencing gel. Since nonspecific nicks within the target sequence increase the background and reduce the resolution of the sequence, it is advisable to use 1-m-long gels or more. The separations on such gels will lead to a greater distance between the bands so that the background, due to the nonspecific degradation products or hybridization-mismatches, will be diluted. For the gel system described in this book an electrophoresis of about 11 hours is optimal for a maximal resolution of the DNA sequence.