Klenow fragment

RNA (5 μg) extracted from wild‐type P. patens and the lines 2b‐1 and P19‐14 was analyzed by agarose gel electrophoresis on a 1% (w/v) formaldehyde gel, blotted onto Amersham Hybond nx nylon membrane (GE Healthcare) and crosslinked with UV light (Sambrook, Fritsch, & Maniatis, 2001 ). Plasmids containing the 2b or P19 suppressor sequences were used as DNA templates for probe amplification, which was done with Dynazyme II DNA polymerase (Thermo Fisher Scientific) with their respective primers (Table S1) according to the manufacturer's instructions. Probe sizes for 2b and P19 were 237 and 251 bp, respectively. The radioactive probes were prepared with 5 μl of template DNA, 13.5 μl nuclease‐free water and 0.5 μl random hexamers (200 ng/μl) and were boiled for 10 min, followed by cooling on ice for 5 min. Klenow buffer (3 μl), 1 μl Klenow fragment, 3 μl of dNTP‐dCTP and 4 μl of [α‐³²P]dCTP (PerkinElmer, Turku, Finland) was added, followed by incubation at 37°C for 1 hr. The probe was purified with the QIAquick Nucleotide Removal kit (Qiagen). Hybridization was carried out at 65°C overnight. The membrane was washed three times with 1× SSC (3.0 M NaCl and 0.3 M sodium citrate), placed into a cassette with an imaging plate and exposed overnight. Radiation energy was scanned and documented with the fluorescent image analyzer FLA‐5100 (Fuji Photo Film Co. Ltd., Tokyo, Japan).