A method for high-concentration agarose gel preparation and its application in high-resolution separation of low-molecular-weight nucleic acids and proteins

Abstract

Separation of nucleic acids and proteins using gels has always been a crucial part of molecular biology research. For low-molecular-weight nucleic acids and proteins, low- and medium-concentration agarose gels cannot achieve the high resolution as polyacrylamide gels. We found that 6 %–14 % high-concentration agarose gels (HAGs) could be easily dissolved in an autoclave and the vertical gel cast can be effortlessly filled using an easy-made plastic box. Coupled with the improved buffer condition, HAG electrophoresis resulted in a good resolution of DNA and protein bands. With conventional TBE buffer plus 0.2 % NaCl, DNA fragments that differ by 2–5-bp within the 50–200-bp size range can be resolved on 6 %–8 % HAGs. By using TBE without NaCl, DNA fragments that differ by 2-bp or 2-nt within the 10–100-bp size range can be well resolved on >8 % HAGs. Using a buffer system comprising 1 M Tris-Cl for gel preparation, 0.2 M Tris-Cl/0.2 % SDS as upper tank buffer, and 0.2 M Tris-Cl as the lower tank buffer, HAGs achieved good molecular weight separation of total bacterial and plant proteins in the 10–200 kDa range. In conclusion, we developed a method for HAG preparation and electrophoresis of low-molecular-weight nucleic acids and proteins.