Vacuum Blotting: a Simple Method for Transferring DNA from Sequencing Gels to Nylon Membranes

We describe a vacuum blotting procedure for transferring DNA fragments from conventional polyacrylamide sequencing gels to nylon membranes. The method employs a combination of vacuum-assisted diffusion (effected by a standard gel drier) and an osmotic gradient (effected by over- and underlying filters presoaked in ammonium acetate). Fragments up to 310 nucleotides in length transfer at 40-60% efficiency within 90 min. When combined with indirect end-labelling, the method allows genomic sequencing of a single-copy gene of Saccharomyces cerevisiae employing as little as 5 micrograms DNA per lane.