Pouring Linear and Buffer-Gradient Sequencing Gels

The products of sequencing reactions are separated on thin, low percentage (usually 6%) polyacrylamide gels. Normally, these gels are 40–50 cm in length, 20 cm wide, and between 0.3 and 0.4 mm thick. Longer gels (to enable more sequence to be read from a single run) up to 100 cm in length can be poured, as can wider gels (to enable more clones to be loaded onto a single gel). However, these larger gels are more difficult to handle after electrophoresis, i.e., during fixation, drying, and auto-radiography. The methods given in this chapter deal only with the “standard” size of sequencing gels, but the principles are the same for larger gels, except that more gel mix will be needed, and different sizes of combs, spacers, and glass plates may also be needed.