α 32p datp

For northern blots, 20 μg of denatured total RNA were loaded on a 1% agarose gel. Electrophoresis, blotting and hybridization were performed as described previously [40 (link)], or using the NorthernMax-Gly Kit (Ambion) as recommended by the supplier. Southern blot hybridization was performed as in [12 (link)]. Electrophoresis of PstI-digested genomic DNA (2 μg per lane) or RT-PCR products were carried out in 0.8%–2% agarose gels (Resolva GQT–for smaller products, Basica LE GQT for larger fragments (Prona)) in 0.5x TBE buffer, and transferred to Hybond N+ or Hybond XL membranes (GE Healthcare) in 0.4 N NaOH. Double-stranded probes were labeled by random priming with [α-³²P] dATP (3000 Ci/mmol, Hartmann Analytic). Oligonucleotide probes were labeled with [γ-³²P] ATP (3000 Ci/mmol, Hartmann Analytic) using T4 polynucleotide kinase. Southern blots were hybridized at 60°C and washed in 0.2x SSC and 0.1% SDS at 60°C prior to image plate exposure. Northern blots were hybridized at 42°C in Ultrahyb buffer (Ambion) and washed as recommended by the supplier. All radioactive signals were quantified using ImageJ. Hybridization probes are described in S1 and S2 Tables.

Genomic DNA prepared from suspension‐cultured cells was digested with XbaI, BamHI/SpeI, or SacI (NEB) and separated on a 0.6% (small donors) or 0.4% (large donors) (w/v) agarose gel at 60 V for 3 hr. Prior to transfer, the DNA was depurinated by incubating the gels in 0.25 M HCl for 15 min. The DNA was subsequently denatured by incubation in 0.5 M NaOH and 0.5 M NaOH/1.5 M NaCl for 30 min each. After neutralization (1 M Tris, 1.5 M NaCl, pH 7.0) for 30 min, the DNA was transferred to a positively charged nylon membrane (Carl Roth) by vacuum transfer with the Vacu‐Blot device according to the manufacturer's instructions (Biometra, Göttingen, Germany) using 2 × SSC. The DNA was immobilized on the membrane by incubation at 80°C for 2 hr. Probes were labeled using α³²P‐dATP (Hartmann Analytic) and the DecaLabel DNA labeling kit (Thermo Fisher Scientific). Hybridization was performed using the Roti‐Hybri‐Quick solution (Carl Roth) according to the manufacturer's instructions. For probe preparation, the following regions were PCR amplified from pDAB113628: 48‐1041 (3′ end of target T‐DNA), 8455‐9491 (5′ end of target T‐DNA), and 9044‐9784 (TR intron). Region 2181‐2891 was PCR amplified from pDAB113676 to prepare the DsRed_5′probe.