Rapid isolation of both double-stranded RNA and PCR-suitable DNA from the obligate biotrophic phytopathogenic fungus Uncinula necator using a commercially available reagent.
A method for rapid extraction of both double-stranded RNA (dsRNA) and DNA from an obligate biotrophic phytopathogenic fungus is described. Lyophilised fungal material is incubated in a commercial guanidium thiocyanate reagent. Proteins and cell debris are centrifuged by chloroform precipitation. After precipitation in isopropanol and washing in 75% ethanol, nucleic acids are resuspended in water (10 microl/mg fungal dry weight). DsRNA is directly visualised by agarose gel electrophoresis. DNA contained in 10-fold dilutions of the samples proved to be suitable for PCR-based experiments.
Unité de Recherches Intégrées sur la Vigne, Institut National de la Recherche Agronomique, Villenave d'Ornon, France. delye@bordeaux.inra.fr
C
C
Délye
M F
MF
Corio-Costet
D001203:Ascomycota / Q000235:genetics / Q000302:isolation & purification / Q000821:virology*; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006146:Guanidines; D016133:Polymerase Chain Reaction / Q000379:methods*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D019105:Random Amplified Polymorphic DNA Technique; D019685:Rosales / Q000382:microbiology; D013861:Thiocyanates
D004271:DNA, Fungal; D006146:Guanidines; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D013861:Thiocyanates; C054436:guanidine thiocyanate