A rapid, high resolution DNA sequencing gel system.
We have developed a simple method which significantly increases the efficiency of manual DNA sequencing. This method increases both the ease of gel preparation and the quality of fragment resolution. Our system involves (i) casting of gels horizontally, without sealing around the plates; (ii) the use of a self-forming buffer gradient to stack bands in the lower part of the gel; (iii) separation of the samples on two 0.2-mm-thick acrylamide gels (4.5 and 4%) with overlapping readings; (iv) "nonsmiling" electrophoresis with very simple, self-made electrophoresis stands; and (v) prior to exposure in situ dry fixation of the gel matrix to the glass support without previous covalent binding of the gel. On average, we are able to read from nucleotide position 50 to position 600 without ambiguity.
Lang
B F
BF
Université de Montréal, Département de Biochimie, Quebec, Canada.
Burger
G
G
D000178:Acrylamides; D001345:Autoradiography; D004247:DNA / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D009710:Nucleotide Mapping / Q000379:methods*; D013462:Sulfur Radioisotopes
D000178:Acrylamides; D007202:Indicators and Reagents; D013462:Sulfur Radioisotopes; D004247:DNA