Nucleospin extract 2 kit

Total plant DNA was extracted from leaf tissue using a cetyltrimethylammonium bromide-based method (Doyle and Doyle, 1990 ). Isolated total DNA was digested overnight with HindIII and separated by gel electrophoresis in a 0.8% agarose gel. Subsequently, DNA fragments were transferred onto Hybond XL membranes (GE Healthcare, Little Chalfont, UK). A 550bp amplicon of the psaB coding region was generated using primer pair 7247 (5′ CCCAGAAAGAGGCTGGCCC 3′) and 7244 (5′ CCCAAGGGGCGGGAACTGC 3′). The PCR product was purified from gel slices using the Nucleospin Extract II kit (Machery-Nagel, Düren, Germany) and used as a template to generate the restriction fragment length polymorphism (RFLP) probe using the MegaPrime DNA Labeling System (GE Healthcare) with ³²P-dCTP. For northern blot analysis, total cellular RNA was isolated using the peqGOLD TriFast reagent (Peqlab GmbH, Erlangen, Germany) according to the manufacturer’s specifications. RNA samples were separated in 1% agarose gels under denaturing conditions and transferred to Hybond XL membranes. The coding sequences of CYP79A1, CYP71E1, and UGT85B1 were isolated by restriction digestion from the transformation vector and purified with the Nucleospin Extract II kit (Machery-Nagel). The isolated DNA was radiolabelled by random priming with ³²P-dCTP using the MegaPrime DNA Labeling System (GE Healthcare).

Reactions for plasmids were 10 μl with 0.77 pmol (77 nM) of R.PabI, 0.11 pmol (11 nM) of plasmid DNA in 0.1 M sodium phosphate buffer (pH 6.5), incubated at 37°C for 30 min. As indicated, reaction mixtures were treated with 5 μg/ml proteinase K in10 mM EDTA and 0.1% sodium dodecyl sulfate (SDS) overnight at 37°C and incubated at 85°C for 6 h (experiments in Figure 4B). In experiments with oligonucleotides, 1 pmol (100 nM) of a double-stranded oligonucleotide substrate was reacted with 9.2 pmol (920 nM) of R.PabI in 10 μl of 0.1 M sodium phosphate buffer (pH 6.5) at 37°C for 3 h and purified with NucleoSpin Extract II Kits (MACHEREY-NAGEL). Half the reaction mixtures were treated with 0.1 M N,N′- dimethylethylenediamine (DMED) at 37°C for 1 h to cleave DNA at AP sites. Products were separated through 10% Long Ranger polyacrylamide gels (TaKaRa) by 50 V for 2 h (experiments in Figure 2C).
Alternatively, 0.2 pmol (10 nM) of a double-stranded oligonucleotide substrate containing the 5′-GTAC sequence and a 5′-³²P label on either strand was incubated with 0–0.5 pmol (0–25 nM) of R.PabI in 0.1 M phosphate buffer (pH 6.5, 20 μl) at 70°C for 1 h. Half the reaction mixture was treated with 0.1 M NaOH at 70°C for 10 min to cleave DNA at AP sites and neutralized with HCl. Samples were separated by 18% denaturing PAGE (experiments in Figure 3).