Purification of Cucumber Pale Fruit Viroid

Abstract

Cucumber pale fruit viroid was purified from infected cucumber leaves and stems. Nucleic acids were extracted from the frozen tissue by phenol-CHCl₃-SDS, and precipitated with ethanol. Polysaccharides were removed by ethylene glycol monomethyl ether treatment and phenolic substrances by cetyltrimethyl ammonium bromide. After 2M LiCl soluble fraction was treated with DNase, low molecular weight RNAs were obtained. The viroid was further purified by CF-11 cellulose chromatography and 15% polyacrylamide gel electrophoresis. Yield of the purified viroid was about 3-6μg/200g of tissue. Five percent polyacrylamide gel electrophoresis of the purified viroid in the presence of urea revealed two bands both associated with infectivity.