Probe DNA was prepared as described in our previous report [26] (link). Genomic DNAs of wild-type (C515), C515-SpA1, C515-SpA2, and C515-EGFP silkworms were prepared from adult moths as follows. First, the adult body was homogenized in 2 mL of homogenization buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, 1% SDS and 0.2 mg mL–1 proteinase K); the mixtures were shaken for 1 h at 50°C. After being shaken, the homogenates were extracted once with TE-saturated phenol, once with TE-saturated phenol–chloroform–isoamyl alcohol, and then precipitated with ethanol.
About 5 µg of each genomic DNA were digested with KpnI or HindIII. Digested DNA (2 µg) was applied to 0.8% agarose gel electrophoresis and then blotted onto a Hybond N+ membrane (GE Healthcare). The piggyBac R-arm sequence was then detected with an AlkPhos Direct Labeling and Detection Kit (GE Healthcare), using the PCR-amplified piggyBac R-arm sequence [26] (link), in accordance with the manufacturer's instructions. Signals were detected with an LAS-3000mini compact chemiluminescence system (Fujifilm Corporation).
About 5 µg of each genomic DNA were digested with KpnI or HindIII. Digested DNA (2 µg) was applied to 0.8% agarose gel electrophoresis and then blotted onto a Hybond N+ membrane (GE Healthcare). The piggyBac R-arm sequence was then detected with an AlkPhos Direct Labeling and Detection Kit (GE Healthcare), using the PCR-amplified piggyBac R-arm sequence [26] (link), in accordance with the manufacturer's instructions. Signals were detected with an LAS-3000mini compact chemiluminescence system (Fujifilm Corporation).