Author: Mouzakis, Kathryn D.; Lang, Andrew L.; Vander Meulen, Kirk A.; Easterday, Preston D.; Butcher, Samuel E.
Title: HIV-1 frameshift efficiency is primarily determined by the stability of base pairs positioned at the mRNA entrance channel of the ribosome Document date: 2012_12_15
ID: ix8du1er_11
Snippet: Microgram quantities of RNA for the frameshift assay were transcribed in vitro using linearized p2luc plasmid DNA, purified His6-tagged T7 RNA polymerase (10Â), 11.25 mM NTPs and two units of RNasin Plus RNase Inhibitor (Promega), in 200 ml for 90 min at 37 C. Pyrophosphate was pelleted by centrifugation (10 min, 13 200 rpm, room temperature) and RNA was phenol/ chloroform extracted. Unincorporated NTPs and salt were separated from the RNA using.....
Document: Microgram quantities of RNA for the frameshift assay were transcribed in vitro using linearized p2luc plasmid DNA, purified His6-tagged T7 RNA polymerase (10Â), 11.25 mM NTPs and two units of RNasin Plus RNase Inhibitor (Promega), in 200 ml for 90 min at 37 C. Pyrophosphate was pelleted by centrifugation (10 min, 13 200 rpm, room temperature) and RNA was phenol/ chloroform extracted. Unincorporated NTPs and salt were separated from the RNA using size-exclusion chromatography [two Econo-Pac P6 cartridges (Bio-Rad) in series]. Monomeric RNA folding was achieved by denaturation at 95 C for 5 min followed by incubation on ice for 30 min. RNAs were lyophilized to dryness and resuspended in water to a concentration of 1 mg/ml and stored in aliquots ($25 ml) at À80 C. RNA integrity and purity were checked with 1% agarose gel electrophoresis. Finally, RNAs used for UV spectroscopy were purchased from IDT.
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