Efficient transfer of highly resolved small DNA fragments from polyacrylamide gels to DBM paper.
A procedure is described that combines high resolution of small DNA molecules (10 to 250 bases) with high transfer efficiency from polyacrylamide gels to diazobenzyloxymethyl (DBM) paper. The DNA fragments are separated electrophoretically in denaturing or nondenaturing step gels. These consist of a short gel of relatively high polyacrylamide concentration (8%) above a long gel of relatively low polyacrylamide concentration (4%). Step gels permit a high resolution of small DNA fragments in gels of sufficiently low polyacrylamide concentration from which efficient transfer to DBM paper is feasible. The combination of the step gel with a short treatment of the gel before transfer ensures a high transfer efficiency. As much as 30% and 50% of the DNA applied to nondenaturing and denaturing gels, respectively, are bound covalently to the DBM-paper. Optimal conditions for hybridization to DBM-linked DNA molecules of 30 to 250 base length are described.
D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D008722:Methods; D008747:Methylcellulose / Q000031:analogs & derivatives; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010209:Paper
C024258:diazobenzyloxymethyl cellulose; D008747:Methylcellulose; D004247:DNA