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.
Gross
D S
DS
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130.
Collins
K W
KW
Hernandez
E M
EM
Garrard
W T
WT
D001345:Autoradiography; D004271:DNA, Fungal / Q000235:genetics* / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting / Q000379:methods*; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D009757:Nylons; D009997:Osmotic Pressure; D015347:RNA Probes; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D008567:Membranes, Artificial; D009757:Nylons; D015347:RNA Probes