Fla 7000 imaging analyzer

RAD51 or DMC1 (0.4 μM) was incubated with the ³²P-labeled 5S 70-mer single-stranded oligonucleotide (1 μM in nucleotides) at 37 °C for 5 min, in a reaction buffer containing 24 mM HEPES (pH 7.5), 1 mM Tris-HCl, 35 mM NaCl, 45 mM KCl, 0.16 mM EDTA, 1 mM DTT, 0.4 mM 2-mercaptoethanol, 2% glycerol, 1 mM MgCl₂, 1 mM ATP, and 100 μg/ml BSA. An aliquot (1 μl) of HOP2-MND1 (final 25 nM) was then added, and the samples were further incubated at 37 °C for 5 min. Subsequently, 1 μl of the naked dsDNA (final 30 μM in nucleotides) or nucleosomal dsDNA (final 30 μM in nucleotides) was added. The reaction mixtures were further incubated at 37 °C for 10 min, and the reactions were stopped by the addition of 2 μl of stop solution, containing SDS (0.2%) and proteinase K (1.4 mg/ml, Roche Applied Science). The resulting DNA products were analyzed by 1% agarose gel electrophoresis, in 1× TAE buffer at 4 V/cm for 2 h. The gels were dried and exposed to an imaging plate. The gel images were visualized using an FLA-7000 imaging analyzer (Fujifilm), and the band intensities were quantitated with the Multi Gauge software (Fujifilm).

Total RNA (2 μg) from cultured cells was dissolved by 10% denaturing PAGE, stained with SYBR Gold (Invitrogen), and blotted onto a nylon membrane (Amersham Hybond N+; GE Healthcare) in 1× TBE using a Transblot Turbo apparatus (Bio-Rad). The membrane was air-dried and irradiated twice with UV light (254 nm, 120 mJ/cm²; CL-1000, UVP) to crosslink the blotted RNA. Hybridization was performed essentially according to the manufacturer’s instructions (PerfectHyb, TOYOBO) at 52 °C with 4 pmol of 5′-³²P-labeled oligonucleotides (Supplementary Data 1) specific for each mt-tRNA. The membrane was washed three times with 1× SSC, dried, and exposed to an imaging plate (BAS-MS2040, Fujifilm). Radioactivity was visualized using an FLA-7000 imaging analyzer (Fujifilm).

Total RNAs (2 µg) from differentiating Hu5/KD3 cells were dissolved by 10% denaturing PAGE, stained with SYBR Gold (Invitrogen), and electroblotted onto a nylon membrane (Amersham Hybond N+; GE Healthcare) in 0.5×TBE using a Transblot Turbo apparatus (Bio-Rad). DNA probes complementary to tRNA^Sec (Supplementary Data 4) were phosphorylated with [γ-³²P] ATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase (Toyobo). The membrane was UV cross-linked and hybridized with 4 pmol of 5′-³²P-radiolabeled DNA probe overnight at 55 °C in PerfectHyb solution (Toyobo). The membrane was washed three times with 1× SSC, dried, and exposed to an imaging plate (BAS-MS2040; Fujifilm). Radioactivity was visualized using an FLA-7000 imaging analyzer (Fujifilm).