For each methyltransferase reaction, 100 ng of refolded tRNA was incubated with 42 nM of purified protein elution along with 50 mM TRIS pH 7.5, 0.1 mM EDTA, 1 mM DTT, 0.5 mM S-adenosylmethionine (NEB) for 4 h at 30 °C. RNA was purified using RNA Clean and Concentrator Zymo-Spin IC Columns where the RNA was resuspended in 6 μl of RNase-free water. To observe if methylation at residue 32 occurred, we utilized primer extension analysis where reverse transcriptase extension would be impeded if methylation occurred. Extracted tRNA was pre-annealed with 0.625 pmol of a 5′-32P-radiolabelled oligonucleotide that hybridizes around 6 nucleotides downstream residue 32 with 2.8 μl 5X hybridization buffer (250 mM TRIS pH 8.5, 300 mM NaCl) to a total of 14 μl. The mixture was heated at 95 °C for 3 minutes and then allowed to slowly cool to either 58 °C. In all, 14 μl of extension mix (0.12 μl of avian myeloblastosis virus reverse transcriptase (Promega), 2.8 μl 5X RT buffer, 1.12 μl 1 mM deoxynucleotidetriphosphates, RNase-free water to 14 μl) was added to each
