UNIT 7.6 Denaturing Gel Electrophoresis for Sequencing
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Slatko, B. E. and Albright, L. M. 2001. Denaturing Gel Electrophoresis for Sequencing. Current Protocols in Molecular Biology. 17:7.6:7.6.1–7.6.13.
- Published Online: 1 MAY 2001
- Published Print: OCT 1991
The accuracy of DNA sequence determination depends largely upon resolution of the sequencing products in denaturing polyacrylamide gels. This unit provides a detailed description of the setup, electrophoresis, and processing of such gels. In general, the gels required for DNA sequencing are 40-cm long, of uniform thickness, and contain 4% to 8% acrylamide and 7 M urea. Modifications of this protocol increase the length of readable sequence information which can be obtained from a single gel (i.e., forming the gel with wedge-shaped spacers to create a field gradient, or incorporating a buffer gradient, an electrolyte gradient, or an acrylamide step gradient into the gel). A modification to the Basic Protocol--inclusion of formamide in the sequencing gel--is designed to overcome gel compressions arising from secondary structure in the sequencing products during gel electrophoresis. A discussion of acrylamide concentrations and electrophoresis conditions is included in the Commentary.