Portal de Periódicos da CAPES

Construction and application of a modified “gene machine”: A circular concentrating preparative gel electrophoresis device employing discontinuous elution

1980; Elsevier BV; Volume: 106; Issue: 2 Linguagem: Inglês

10.1016/0003-2697(80)90548-5

1096-0309

Laura Carreira, Bruce C. Carlton, Steven M. Bobbio, Ronald T. Nagao, Richard B. Meagher,

Gene expression and cancer classification

A modified version of a preparative circular gel electrophoresis apparatus, first described by Edwin Southern (Medical Research Council, University of Edinburgh, Edinburgh, Scotland), has been constructed. The apparatus fractionates a large volume of sample into concentric bands which migrate toward a small circular collection chamber. Samples exiting the gel into the collection chamber are concentrated against a dialysis membrane which encloses the inner electrode and are pumped from this center chamber into a fraction collector at fixed time intervals. The apparatus has been employed to fractionate samples of DNA (10 mg) by electrophoresis through either agarose or acrylamide gels. Two examples of nucleic acids which have been successfully fractionated are given: restriction endonuclease cleavage fragments of total soybean DNA, and a heterogeneous mixture of covalently closed circular plasmid DNA from Bacillus megaterium. Franctionated DNA is suitable for molecular cloning directly from acrylamide and, after one additional treatment, from agarose. The run time for DNA treated with restriction endonuclease is from 24 to 48 h. Purification of 60- to 200-fold is common for a DNA restriction fragment from a total genome.