Rapid isolation of double stranded RNA segments from disulphide crosslinked polyacrylamide gels.
A simple method was developed to isolate dsRNA segments from disulphide crosslinked polyacrylamide gels. The dsRNA preparations from the P4 killer virus strain 77 of Ustilago maydis containing 7 genomic segments with molecular sizes ranging from 0.36 to 6.7 kbp, the 20 kbp dsRNA associated with the '447' cytoplasmic male sterility in Vicia faba and the 23 kbp genomic dsRNA of citrus tristeza virus (CTV) were separated on disulphide crosslinked polyacrylamide gels. After UV visualisation, the dsRNA bands were excised from the gel and dissolved using 2-mercaptoethanol. The dsRNA were then purified from the solubilized fractions by specific adsorption on microgranular cellulose and elution with a small volume of water. The method is rapid, simple and convenient for the isolation of all the tested dsRNAs segments.
S. Tolkowsky Laboratory, Department of Virology, Volcani Center, Bet Dagan, Israel.
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P
Dulieu
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Bar-Joseph
D003432:Cross-Linking Reagents; D003538:Cystamine / Q000031:analogs & derivatives; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010942:Plant Viruses / Q000235:genetics*; D012328:RNA Viruses / Q000235:genetics*; D012330:RNA, Double-Stranded / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification*
D003432:Cross-Linking Reagents; D004220:Disulfides; D012330:RNA, Double-Stranded; D012367:RNA, Viral; C027747:N,N'-bisacrylylcystamine; D003538:Cystamine