Rna cleanup kit

A 263-bp template encoding E. coli 9S RNA was amplified from pKK233-2 (provided by Dr. AJ Carpousis) using PCR (primers are depicted in Table 4). The PCR reaction was carried out with Phire Hot Start II polymerase (Finnzymes) according to the manufacturer’s protocol. Incorporated in one of the primers was a T7 RNA polymerase recognition sequence (underlined). PCR products were checked on a 1% agarose gel and purified with the QIAquick PCR Purification Kit (QIAGEN). The PRC product was used as a template for IVT.
IVT reactions were carried out in 200 μl volumes according to standard protocol. Each reaction was set up with 3.5 μg of template, and additionally supplemented with T7 RNA polymerase and DMSO (3% (v/v)). The IVT reaction was run at 37°C for 5 hr, and TURBO DNase I (Invitrogen) was added in the last 30 min to digest the DNA template. Transcribed 9S rRNA was then gel-purified from a 4% polyacrylamide gel. Bands containing RNA were visualized by UV–shadowing and excised. RNA was recovered from gel slices by overnight electroelution at 100 V in 1×TBE buffer using an EluTrap System (Whatman). Finally, RNA was purified with an RNA cleanup kit (Thermo Fisher Scientific) and the concentration was measured with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Purified 9S rRNA was stored in MilliQ water at –20°C.

Point mutations were introduced into mouse α, β, and γ ENaC plasmid DNAs using QuickChange Ⅱ XL site-directed mutagenesis kit (Agilent). The DNA sequences, including those of wildtype and mutant ENaC, were confirmed by direct sequencing at the Genomics Research Core of University of Pittsburgh. Wildtype and mutant RNAs were made from the linearized DNA using T3 RNA in vitro transcription kit and purified with RNA Cleanup Kit (Thermo Fisher Scientific Inc). RNA quality was verified by denaturing RNA gel analysis, and RNA concentrations were estimated by a spectrophotometer. One mutation (αN510C) was made previously (41 (link)).