Storm 840 phosphorimager system

Southern blot was performed as previously described^{47 (link)}. Briefly, 3 µg of total leaf DNA digested with NcoI (New England Biolabs, Beverly, MA, USA) were electrophoresed in 0.8% agarose gel, and blotted onto HybondN+ Nylon membrane (Amersham Biosciences, Uppsala, Sweden). The membrane was hybridized with a ³²P-labeled trnI/A DNA probe, generated by random priming with a Prime-a-Gene kit (Promega, Madison, WI, USA). Pre-hybridization and hybridization were carried out following published protocols^{47 (link),77} . The blot was analyzed after exposure to a storage phosphor screen, using a Storm 840 PhosphorImager system (Amersham Biosciences).

Total DNA was extracted from leaves as described by Dellaporta et al. (1984) (link). The DNA (2.5 μg) was digested overnight with NcoI enzyme (New England Biolabs, USA), electrophoresed in 0.8% agarose gels and blotted onto Hybond-N+ Nylon membranes (Amersham Biosciences, USA). Specific DNA sequences were detected by hybridization with α-³²P-labeled trnI/A DNA probe. The probe was generated by random priming with a Prime-a-Gene kit (Promega, USA). Pre-hybridization and hybridization were carried out at 65°C in Church’s hybridization solution (Church and Gilbert, 1984 (link)) for 2 and 16 h, respectively. Membranes were washed twice with gentle shaking for 30 min in 0.2X SSC, 0.1% SDS at 65°C. The blot was exposed to a storage phosphor screen, which was analyzed in a Storm 840 PhosphorImager system (Amersham).