Rediprime 2 random prime labeling system

Pre-hybridization and hybridization were performed in hybridization solution in glass tubes (30 cm × 4 cm) at 65°C under continuous rotation in a hybridization oven (Bachofer, Reutlingen, Germany). The pre-hybridization was performed overnight. Upon adding the denatured radio-active probe, the hybridization was performed for at least 16 hours. After hybridization the filter was washed with SSC and SDS solutions. The filter was wrapped in Saran wrap and exposed overnight to a phosphoimager screen (Molecular Dynamics) in a cassette at room temperature. 50–100 ng of gel-purified PCR product for Northern analysis was used. Probe was prepared according to Rediprime II Random Prime Labeling System protocol manual provided by Amersham Biosciences. Probe was later on purified on a Sephadex G25 column.

To analyze the genomic DNA for integration of the J1-1 gene, pepper genomic DNA was isolated using a DNeasy Plant Maxi Kit (Qiagen, Hilden, Germany) as described by the manufacturer. For Southern blot analysis, 15 µg of each DNA sample was digested with EcoRI and separated on a 1.0% (w/v) agarose gel. The digested DNA was then transferred to a nylon membrane and hybridized with HPT or J1-1 gene probes that was labeled with [α³²P] dCTP using the Rediprime II Random Prime Labeling System (Amersham Biosciences, UK). After hybridization, the membranes were exposed at −80°C on Kodak XAR-5 film (Kodak, Rochester, NY) using an intensifying screen.
For Northern blot analysis, total RNA was extracted from the pepper fruits using a RNeasy Plant Kit (Qiagen, Hilden, Germany). 10 µg of the total RNA was separated on 1.2% denaturing agarose gels and blotted onto a Hybond N⁺ membrane (GE Healthcare, Buchinghamshire, UK). The blots were then hybridized with [α³²P] dCTP-labeled respective probes that were amplified by PCR. The primers used for probes were shown in Table S1.