Dig oligonucleotide tailing kit

In situ hybridization of frozen sections of Lymnaea CNS was performed as previously described^{23 (link)}. The labelled probe (5′-CACAGGA(AC)GGTATGGTGTTCT-3′) was prepared using the DIG Oligonucleotide Tailing Kit (Roche) according to the manufacturer’s protocol.

Northern blot analysis was performed essentially as previously described19 (link). Total RNAs were extracted from leaf tissues using TRIzol reagent (Invitrogen) and treated with DNase (TAKARA). 3 μg of total RNAs were separated on a denaturing 2% formaldehyde agarose gel and transferred to Hybond-N+ membranes (Amersham Bioscience) using 20 × SSC. The RNAs were cross-linked to the membrane matrix by UV for 45 s. Northern blotting for assays of CWMV genomic RNAs were carried out using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). DNA oligonucleotides complementary to the 3′-terminus of the CWMV genome or specifically complementary to CWMV RNA1 were labeled with digoxigenin (DIG) at their 3′ ends using the DIG Oligonucleotide Tailing Kit (Roche) and then purified using a G25 Sephadex column (GE). Membranes were prehybridized for 2 h and hybridized overnight at 42 °C using the DIG Luminescent Detection Starter Kit for Nucleic Acids (Roche). The hybridization signals were visualized by the Amersham Imager 600 (GE). All these procedures followed the manufacturers’ instructions.

Total mitochondrial RNAs (mtRNAs) were obtained from mitochondria isolated from mutant and control cell lines (∼2.0 × 10⁸ cells) using a ToTALLY RNA kit (Ambion), as described previously (31 (link)). Two micrograms of total mitochondrial RNA was electrophoresed through a 10% polyacrylamide–7 M urea gel in Tris-borate-EDTA (TBE) buffer (after heating the sample at 65°C for 10 min) and then electroblotted onto a positively charged nylon membrane (Roche) for hybridization analysis with oligodeoxynucleotide probes. For the detection of tRNA^Ala, tRNA^Lys, tRNA^His, and tRNA^Glu, nonradioactive digoxigenin (DIG)-labeled oligodeoxynucleotides specific to each RNA and 5S RNA were as detailed previously (9 (link), 15 (link), 32 (link)– (link)34 (link)). DIG-labeled oligodeoxynucleotides were generated by using a DIG oligonucleotide tailing kit (Roche). The hybridization was carried out as detailed elsewhere (9 (link), 15 (link), 32 (link)– (link)34 (link)). Quantification of density in each band was done as detailed previously (9 (link), 15 (link), 32 (link)– (link)34 (link)).