Denaturing Polyacrylamide Gel Electrophoresis

Thin polyacrylamide gels that contain a high concentration of urea as a denaturant are capable of resolving short (<500 nucleotides) single-stranded fragments of DNA or RNA that differ in length by as little as one nucleotide. Such gels are uniquely suited for nucleic acid sequence analysis, which is required, for instance, for all footprinting protocols. Thicker gels are often used to purify oligonucleotides. This appendix describes the pouring, running, and processing of a typical sequencing gel, which is 40 cm long with a uniform thickness of 0.4 mm, containing 7 M urea and 4% to 8% acrylamide.