Genelute pcr clean up kit

S. cerevisiae tRNA^Phe_GUG (phenylalanine anticodon GAA was replaced with the histidine anticodon GUG) and its 5′-truncated tRNA fragments (template RNAs) were transcribed using T7 RNA polymerase. Double-stranded DNAs encoding the T7 promoter and target RNA sequences were amplified by PCR with three overlapping primers, and cloned into the BamHI/HindIII site of pUC19. The inserted sequences were verified by DNA sequencing. Double-stranded DNA transcription templates were obtained by PCR and purified using a GenElute PCR Clean-Up kit (Sigma). The in vitro transcription was performed using a DuraScribe T7 Transcription kit (Epicentre) at 37°C for 6 h. The reaction mixture was subsequently treated with DNase I at 37°C for 30 min to degrade the template DNA, and purified using 10% denaturing urea-polyacrylamide gel electrophoresis (Urea-PAGE). RNAs were extracted from gel slices and refolded simultaneously in H₂O at 4°C for 18 h. The extracted RNA samples were precipitated with ethanol, dissolved in TE buffer pH8.0, and stored at −80°C.

PCR amplification with Phusion Hot Start II high-fidelity polymerase (Thermo Scientific, Waltham, MA) was performed according to the manufacturer’s manual using high-performance liquid chromatography (HPLC)- or PAGE-purified, custom-synthesized oligonucleotide primers (Sigma-Aldrich). Diagnostic PCR was done with DreamTaq (Thermo Scientific) and desalted primers (Sigma-Aldrich). DNA fragments obtained by PCR were loaded on gels containing 1% or 2% (wt/vol) agarose (Thermo Scientific) and 1× Tris-acetate-EDTA buffer (Thermo Scientific), excised, and purified (Zymoclean, D2004; Zymo Research, Irvine, CA). Alternatively, fragments were purified using the GenElute PCR Cleanup kit (Sigma-Aldrich). Plasmids were isolated from E. coli with the Sigma GenElute Plasmid kit (Sigma-Aldrich) according to the supplier’s manual. Yeast plasmids were isolated according to the methods described in reference 50 (link). Yeast genomic DNA was isolated using a YeaStar genomic DNA kit (Zymo Research). E. coli DH5α (18258-012; Invitrogen) was transformed chemically (T3001; Zymo Research) or by electroporation. Chemical transformation was done according to the supplier’s instructions. Electroporation was done in a 2-mm cuvette (165-2086; Bio-Rad, Hercules, CA) by using a Gene PulserXcell electroporation system (Bio-Rad), following the manufacturer’s protocol.

All tethered particle motion and pull-down bridging assay experiments were performed using a random, AT-rich, 685 bp (32% GC) DNA substrate. The DNA substrate was generated by PCR and the products were purified using a GenElute PCR Clean-up kit (Sigma-Aldrich). If required, DNA was ³²P-labeled as described previously (20 (link)). For the electrophoretic mobility shift assay an AT-rich 200 bp (32%GC) was generated using the same procedure.
The oligonucleotides to generate the 12 bp DNA duplex d(CGCATATATGCG) were purchased from Sigma Aldrich. The lyophilized oligonucleotides were solubilized in 20 mM Bis–Tris Buffer, 50 mM or 300 mM KCl, pH 6, combined and hybridized by heating to 98°C for 3 min, followed by slow cooling down to room temperature. The 12 bp DNA duplex was used for NMR titration of the MvaT dimer and DBD.

The yeasts were characterized according to their cell morphology followed by PCR-RFLP patterns of the ITS-rDNA region using the restriction enzyme MspI (New England Biolabs, Ipswich, MA). The strains with identical RFLP banding patterns were grouped and initially considered to belong to the same species. At least one representative strain from each grouping was selected and subjected to sequence analysis of the partial large subunit (LSU) rRNA gene. Genomic DNA of yeasts was isolated using a standard protocol [14 ]. PCR amplification of the LSU rRNA gene was carried out using standard primers and PCR cycling conditions [15 (link)]. The PCR products were purified using a GenElute PCR Clean-Up Kit (Sigma-Aldrich, St. Louis, MO) and sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA). Phylogenetic analyses were made with MEGA 7 [16 (link)] using the Maximum Likelihood algorithm and the Tamura–Nei evolutionary model, as suggested by the implemented model test.