Hybond n nylon transfer membrane

Total RNA (500 ng) was separated on a 7 M urea 6% denaturing polyacrylamide gel and transferred onto a Hybond-N + nylon transfer membrane (GE Life Sciences) then fixed by cross-linking in a UV Stratalinker 2400 using the auto-crosslink button twice. The blots were hybridized at 60 °C for 12 h in 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS), and 10× Denhardt’s solution with ³²P-end labeled probes specific for the full-length tRNA transcript (72–76 nucleotides; Supplementary Data 1 and 2). After two washes (each) in 2× SSC, 0.1% SDS for 20 min at room temperature and 0.5× SSC, 0.1% SDS for 60 min at 60 °C, the membrane was exposed to a storage phosphor screen for 15 min and analyzed on an Amersham Typhoon.

Total RNA (10 µg) was separated on a 4% polyacrylamide (29:1) gel containing 8 M urea at 20 W for 1 h and then transferred to a Hybond-N⁺ nylon transfer membrane (GE Healthcare, Cat. No: RPN303B) at 400 mA for 1 h in 0.5X TBE buffer. RNA was cross-linked to the membrane in a Stratalinker (Stratagene, 120 mJ). The blot was prehybridized in Church buffer at 65 °C (for hTR) or at 42 °C (for oligonucleotide probe) for an hour. Hybridizations with radiolabelled probes were performed in Church buffer at 65 °C (for hTR, probes were generated by nick translation of a polymerase chain reaction (PCR) fragment with ³²P-α-dCTP) and 42 °C (for oligonucleotide probe against 7SL, which was labelled with ³²P-γ-ATP by T4 PNK kinase). The oligonucleotide sequences are listed in Supplementary Table 6. All blots derive from the same experiment and were processed in parallel.