Rediprime 2 dna labeling system

Genomic DNA was isolated using two different methods depending on experimental purpose. Genomic DNA for double-joint PCR and Southern blot was isolated according to a standard protocol (Sambrook and Russell, 2001 ). Genomic DNA used for gene deletion mutant screening by PCR was prepared using a quick extraction method (Chi et al., 2009b ). Gene knockout constructs were produced using double-joint PCR (Yu et al., 2004 (link)), which fuses three different DNA fragments (two 1 to 1.5 kb fragments corresponding to the 5′- and 3′-flank regions of each target gene and a 2.1 kb fragment containing the Hygromycin B phosphotransferase gene) (Table 2). Resulting constructs were introduced into protoplasts of KJ201 as previously described (Sweigard et al., 1992 (link)). Hygromycin B-resistant transformants were screened first using PCR and confirmed by Southern analysis (Sambrook and Russell, 2001 ) (Fig. 2). Hybridization probes were prepared using the Rediprime^™ II DNA Labeling System (GE Healthcare, USA) according to the manufacturer’s instructions, and signals on the membranes were detected using Phosphorimager (BAS-2040, Fuji Photo Film, Japan). For genetic complementation, an intact copy of the mutated gene was amplified by PCR and introduced into protoplasts of individual mutants using a geneticin-resistance gene as a selection marker.

Southern blot analysis was carried out to determine the copy number of inserted T-DNA in the genome of X. grammica. 1 µg of genomic DNA from the wild-type strain and individual transformants was digested with HindIII and was resolved via gel electrophoresis using 0.8% agarose gel at 50 V for 4 h in 0.5% Tris-Acetate-EDTA buffer. Separated DNA fragments were transferred to Hybond N⁺ membrane (Amersham International, Little Chalfont, England) using 10 × SSC (1.5 M NaCl and 0.15 M Sodium citrate) and UV crosslinked.
A 600 bp probe corresponding to the hph gene was obtained by PCR amplification using pBHt2 [21 (link)] as a template and primers HygB_F and HygB_R (Table 1), and the resulting fragment was labeled using ³²P-dCTP via random priming (Rediprime II DNA labeling system, GE Healthcare Life Sciences). Hybridization was carried out overnight at 65 °C in 6 × SSPE (1 × SSPE: 0.18 M NaCl, 1 mM EDTA, and 10 mM sodium phosphate at pH 7.4) containing 1% sodium dodecyl sulfate (SDS) and 100 μg of denatured salmon sperm DNA per mL. Hybridized blots were washed twice in 2 × SSPE and 0.1% SDS for 5 min at 65 °C. Signals were detected using autoradiography films and BAS-MS imaging plate (Fuji Film).

Cells grown under iron-replete conditions (time point 0) were washed two times with iron-free medium and further grown under iron depletion for 24 and 48 h (time points 24 and 48). RNA extraction, blotting, and hybridization were performed as described previously (61 (link)), and RNA was hybridized to a radioactively labeled isiA DNA probe (including the 5′ UTR region) using the Rediprime II DNA labeling system (GE Healthcare Life Sciences). As a control, the same blot was hybridized with a riboprobe against the rnpB RNA using the T7 polymerase Maxiscript kit (Ambion). Primers used to generate the isiA (P16 and P17), rnpB (P18 and P19), and isiB (P20 and P21) probes are given in SI Appendix, Table S2.