Cloning of Four Plant Viruses from Small Quantities of Double-Stranded RNA

The first strand and second strand synthesis were basically that of Gubler and Hoffman with the omission of mung bean nuclease digestion. After second strand synthesis, the DNA was size-fractionated on a Sepharose CL-4B column, C-tailed, annealed to G-tailed pBR322/pUC9, and Escherichia coli DH1/DH5a cells transformed. No differences in length of cDNA and size of inserts was observed when heat- and DMSO-denaturation were used. However, use of MeHg yielded greater incorporation of 32 P-dATP into first strand when compared to the boiling method of denaturation