S. cerevisiae tRNAAlaUGC was in vitro–transcribed by T7 RNA polymerase from a linearized pUC19 vector harboring a corresponding tRNA sequence under the control of a T7 promoter. Transcription took place overnight at 37°C in 20 mM tris-HCl (pH 8.0), 150 mM NaCl, 8 mM MgCl2, 2 mM spermidine, template DNA (100 μg/ml), RNasin (25 mg/ml), pyrophosphatase (0.001 U/ml), and T7 RNA polymerase. To modify tRNA internally with Cy5 dye during transcription, 20% of cytidine triphosphate was substituted with 5-propargylamino-cytidine-5′-triphosphate labeled with Cy5 (Jena Bioscience). Deoxyribonuclease (3 to 10 U; Thermo Fisher Scientific) was added for 30 min at 37°C to remove template DNA followed by purification on HiTrap DEAE FF column (GE Healthcare). tRNA eluate was precipitated overnight at −20°C with 0.3 M sodium acetate (pH 5.5) and 75% ethanol. After centrifugation for 30 min at 4°C (20,000g), the tRNA pellet was washed with 75% ethanol; dried and suspended in 20 mM Hepes (pH 7.5), 50 mM NaCl, and 50 mM KCl; and annealed by gradual cooling from 80° to 40°C at a speed of 0.5°C/min. After annealing, tRNA was supplemented with 1 mM MgCl2 and 1 mM DTT and purified on a Superdex 75 Increase 10/300 GL column (GE Healthcare).

Wild-type bulk tRNA and bulk Δelp3 tRNA were purified from UMY2893 and UMY2916 strains, respectively. Cells were grown in yeast extract/peptone/dextrose (YPD) medium to an OD600 (optical density at 600 nm) of 1.0 to 1.5, collected by centrifugation, and suspended in equal volume of 10 mM tris-HCl (pH 7.5), 1 mM EDTA, 100 mM sodium acetate, and 1% SDS. After freezing in liquid nitrogen, cells were cryo-milled and thawed at 4°C with agitation. The lysate was mixed with equal volume of TRIzol (Thermo Fisher Scientific) and incubated at room temperature for 20 min. Next, 20% (v/v) of chloroform was added, mixed, and incubated at room temperature for 15 min. After centrifugation at 4°C with 3000g, the aqueous phase was collected and further centrifuged at 4°C for another 60 min at 12,000g. Large RNA from the supernatant was precipitated by incubation at −20°C for 3 hours with 33% volume of 8 M lithium chloride and centrifugation at 4°C with 4000g (30 min). tRNA was precipitated as described above and suspended in 4 ml of 10 mM sodium acetate (pH 5.2). tRNA was purified on HiLoad 26/600 Superdex 200 pg (GE Healthcare). Both transcribed and bulk tRNAs were concentrated using an Amicon Ultra-0.5 3k concentrator (Merck Millipore), aliquoted, and stored at −20°C.

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