Rapid alkaline blot-transfer of viral dsRNAs.
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.
D001820:Bluetongue virus / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D009757:Nylons; D012330:RNA, Double-Stranded / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis*; D012087:Reoviridae / Q000235:genetics; D012972:Sodium Hydroxide
D008567:Membranes, Artificial; D009757:Nylons; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012972:Sodium Hydroxide