Northern blot analysis of aminoacylated tRNAs

tRNA extracts containing the tRNA of interest were ethanol-precipitated (no oxidation was performed) and then resuspended in buffer D to a final concentration of 1 µg/µl, as assessed by nanodrop. 2 µg of the tRNA sample was run for 4.5 h at 4°C on a 15 cm long acidic urea PAGE (6.5% acrylamide 19:1, 100 mM NaOAc pH 5, 8 M urea, 0.1% TEMED and 0.1% (NH₄)₂S₂O₈) using 300 mM NaOAc pH 5 as running buffer and 12 W of constant power (~140 V, ~85 mA). The composition of the loading dye (2x) was 100 mM NaOAc pH 5, 8 M urea, 0.1% xylene cyanol FF and 0.1% bromophenol blue.

After electrophoresis, the gel was stained with SYBR Gold to identify the position of tRNAs. An appropriate section of the gel was cut out and transferred onto nylon membrane (Ambion^® BrightStar^®-Plus Positively Charged Nylon Membrane) using the iBlot™ DNA Transfer Stack for the iBlot^® Dry Blotting System (the membrane contained in the transfer stack is replaced). The tRNAs were cross-linked to the membrane (Stratalinker^® UV Crosslinker 2400), which was later immersed for 20 min in Ambion^® ULTRAhyb^®-Oligo buffer for blocking. The biotinylated DNA probe was then added to a final concentration of 3 ng/µl and hybridised overnight at 37°C (tRNA^Pyl probe: 5’-TGGCGGAAACCCCGGGAATCTAACCCGGCT-3’). Excess probe was washed off using 2xSSC buffer and then blotting was then performed with the Thermo Scientific Pierce Chemiluminescent Nucleic Acid Detection Module.