Rapid transfer of DNA from agarose gels to nylon membranes - PubMed (original) (raw)

Rapid transfer of DNA from agarose gels to nylon membranes

K C Reed et al. Nucleic Acids Res. 1985.

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Abstract

The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials afforded by this simple modification is accompanied by a marked improvement in resolution and a ten-fold increase in sensitivity of subsequent hybridization analyses. In addition, we have found that nylon membrane completely retains native (and denatured) DNA in transfer solvents of low ionic strength (including distilled water), although quantitative elution of DNA from the gel is limited to fragments smaller than 4 Kb. This property can be utilized in the direct electrophoretic transfer of native restriction fragments from polyacrylamide gels. Exposure of DNA to ultraviolet light, either in the gel or following transfer to nylon membrane, reduces its ability to hybridize.

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References

    1. Cell. 1975 Jul;5(3):301-10 - PubMed
    1. Gene. 1984 Dec;32(3):409-17 - PubMed
    1. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961-5 - PubMed
    1. J Mol Biol. 1977 Jun 15;113(1):237-51 - PubMed
    1. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835-8 - PubMed

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