Superscript 2 rnase h reverse transcriptase

Oligonucleotides were either purchased from IDT (Coralville, IA) or prepared by solid-phase synthesis using an Expedite 8909 DNA/RNA synthesizer, with reagents and nucleoside phosphoramidites purchased from Glen Research (Sterling, VA), except L-2′-tert-butyldimethylsilyl phosphoramidites, which were from ChemGenes (Wilmington, MA). For coupling of degenerate nucleotides (N), the concentration ratios of the four nucleoside phosphoramidites A : T : G : C were 3.0 : 2.0 : 2.3 : 2.5, respectively, to achieve equal coupling efficiencies. All oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis (PAGE) and desalted by ethanol precipitation. Histidine-tagged T7 RNA polymerase was purified from E. coli strain BL21 containing plasmid pBH161 (provided by William McAllister, State University of New York, Brooklyn). Thermus aquaticus (Taq) DNA polymerase was cloned from total genomic DNA and prepared as described previously.^{18 (link)} Superscript II RNase H⁻ reverse transcriptase, Turbo DNase, and streptavidin-coated magnetic beads (Dynabeads, My-One Streptavidin C1) were from Life Technologies (Carlsbad, CA). Full-length human Dicer protein was provided by Ian MacRae (The Scripps Research Institute, La Jolla, CA). Nucleoside and deoxynucleoside 5′-triphosphates were purchased from Sigma-Aldrich (St. Louis, MO) and [γ-³²P]ATP was from Perkin Elmer (Waltham, MA).