Vacuum blotting: a simple method for transferring DNA from sequencing gels to nylon membranes

David S. Gross; Kerry W. Collins; Efrem M. Hernandez; William T. Garrard

doi:10.1016/0378-1119(88)90168-0

Vacuum blotting: a simple method for transferring DNA from sequencing gels to nylon membranes

David S. Gross, Kerry W. Collins, Efrem M. Hernandez, William T. Garrard

Molecular Biology

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

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 μg DNA per lane.

Original language	English (US)
Pages (from-to)	347-356
Number of pages	10
Journal	Gene
Volume	74
Issue number	2
DOIs	https://doi.org/10.1016/0378-1119(88)90168-0
State	Published - Dec 30 1988

Keywords

Genomic sequencing
RNA hybridization probes
in vivo footprinting
indirect end-labelling

ASJC Scopus subject areas

Genetics

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10.1016/0378-1119(88)90168-0

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title = "Vacuum blotting: a simple method for transferring DNA from sequencing gels to nylon membranes",

abstract = "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 μg DNA per lane.",

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AU - Gross, David S.

AU - Collins, Kerry W.

AU - Hernandez, Efrem M.

AU - Garrard, William T.

PY - 1988/12/30

Y1 - 1988/12/30

N2 - 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 μg DNA per lane.

AB - 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 μg DNA per lane.

KW - Genomic sequencing

KW - RNA hybridization probes

KW - in vivo footprinting

KW - indirect end-labelling

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Vacuum blotting: a simple method for transferring DNA from sequencing gels to nylon membranes

Abstract

Keywords

ASJC Scopus subject areas

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