Digoxigenin high prime dna labeling and detection starter kit 2

To estimate the number of copies of T-DNA fragments in the Ri-21 and Ri-27 lines, 15 μg genomic DNA was digested with HindIII restriction endonuclease, which did not cut within the T-DNA fragment. The digested DNA was fractionated on a 0.8% (w/v) agarose gel, blotted onto nylon membranes, and cross-linked (Sabelli 2007 ). The membranes were then hybridized with a 768-bp specific digoxigenin-labeled Hyg DNA fragment (Additional file 9: Table S2). Probe labeling and hybridization were conducted using a Digoxigenin-High Prime DNA Labeling and Detection Starter Kit II (Roche, Basel, Switzerland).

The hygromycin resistance gene (HygR) served as a probe (length: 858 bp) for Southern blotting. It was amplified by PCR (Table S2) and labeled using the digoxigenin High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics GmbH, Mannheim, Germany). A VP2-specific probe (length: 1268 bp) was created similarly and served as a control. Total DNA of N. oceanica was isolated according to Varela-Álvarez et al. (2006 (link)), and 3 µg was digested with 30 units of HindIII or SacI for 10 h and separated on a 0.8% (w/v) agarose gel. Digested plasmid DNA of pNoc ox Pro(EF)::VP2 (6 ng) and genomic DNA of the wild type served as positive and negative controls, respectively. After depurination, denaturation, and neutralization, the DNA was transferred to a positively charged nylon 6.6 membrane (Biodyne B Membrane, pore size 0.45 μm; Pall Life Sciences, Portsmouth, USA). The membrane was hybridized with the probe overnight at 52 °C. After a washing step, the membrane was blocked and incubated with polyclonal anti-digoxigenin antibodies conjugated to alkaline phosphatase, following the manufacturer’s instructions. Alkaline phosphatase was detected by the chemiluminescence reagent “CSPD ready-to-use” (Roche Diagnostics GmbH), using a CCD camera system (LAS-3000; Fujifilm, Tokyo, Japan).