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Rapid alkaline blot-transfer of viral dsRNAs

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
163
Issue
1
Identifiers
DOI: 10.1016/0003-2697(87)90115-1
Keywords
  • Northern Blots
  • Alkaline Transfer Medium
  • Viral Dsrnas
  • Hybridization
Disciplines
  • Biology

Abstract

Abstract The double-stranded genomic RNAs of reovirus and bluetongue virus can be transferred very efficiently from either sodium dodecyl sulfate-polyacrylamide gels or NuSieve agarose gels onto several nylon membranes. After a brief acid depurination treatment, viral dsRNAs from the gels are transferred at room temperature using 0.2 n NaOH as the transfer medium. Four blots can be obtained within 1 h and each blot contains 15–20% of the input RNA sample. These blots can be used immediately without baking in vacuo. Less than 5% of the “fixed” dsRNAs are removed after repeated washings of the membrane blots. As little as 10 pg of the genomic dsRNA segment can be detected in this alkaline Northern blot. A 20- to 50-fold increase in resolution and sensitivity over traditional Northern blots is routinely achieved. These alkaline blots can be reused 6–10 times after appropriate strip washing and proper handling.

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