N nylon membrane

Genomic DNA was extracted and purified from tissues of transgenic calf using a Wizard^®Genomic DNA Purification Kit (Promega, WI, USA). Then, genomic DNA (20 mg) were digested with EcoR I and Bam HI (Promega, WI, USA), separated by electrophoresis in 1.0% agarose gel and transferred to a N+ nylon membrane (Amersham). A 2.7-kb probe (50 ng) from EGFP and Neo gene-coding sequence was prepared by digesting the pEGFP-IRES-Neo vector with EcoR I and BamH I. Probes were labeled with α³²P dCTP (3000 Ci/mol) using a random primer DNA labeling kit (Pharmacia Biotech) according to the manufacturer’s instructions. Hybridization was carried out following standard procedures.

The CeuI fragments were separated by a CHEF-DR II electrophoresis system (Bio-Rad). Electrophoresis was performed on a 1% agarose (Seakem Gold agarose, BioWhittaker Molecular Applications) gel and 0.5X TBE buffer (Bio-Rad) at 11°C. The electrophoresis conditions were divided into two stages to separate the DNA fragments of various sizes: First stage, pulse time ramped from 6.75 s to 2 min for 20 h at 4 V cm^-1 and in a second stage, pulse time ramped from 0.22 to 5.10 s for 15 h at 6 V cm^-1.
The agarose gels were radiated with UV light for 1 min in a UV Crosslinker (UVP) to fix the DNA. The gels were washed in 250 mM HCl solution for 15 min with moderate shaking. Next, the gels were washed in denaturing buffer (1.5 M NaCl, 0.5 M NaOH) for 2 h and later washed in a neutralization buffer (0.5 M Tris/HCl, 1.5 M NaCl; pH 8.0) for 2 h. The DNA fragments were then transferred onto N+nylon membrane (Amersham Biosciences) via Southern blotting as described previously (Sambrook et al., 1989 ). Finally, the membrane was radiated with UV light to fix the DNA (1 min; UV Crosslinker, UVP).

Genomic DNA was isolated from the Basta tolerant and untransformed control plants using the method of Zidani et al [56] . PCR analysis was carried out using the primers; 5′-GCC CAT GGA GAC CAT TGC TAG ATT TGA TGA TT- 3′ and 5′ -GCG GAT CCT CAC CGA CGC CGG TGA GAG GGT TTA G – 3′ for BjNPR1, and 5′- CTA CCA TGA GCC CAG AAC G – 3′ and 5′- TCA GAT CTC GGT GAC GGG -3′ for bar-nos. DNA isolated from the untransformed control plants was used as the negative control and Ti super binary vector was used as the positive control. For Southern blot analysis, 10 μg of genomic DNA was digested with BglII, electrophoresed on a 0.8% agarose gel and subsequently transferred to an N⁺ Nylon membrane (Amersham Biosciences) and fixed by exposing to UV (1200 μJ for 60 s) in an UV cross linker (Sambrook and Russell, 2001). A 550 bp BjNPR1 internal sequence was labeled with α-³²P dCTP employing ready to go random primer DNA labelling kit (Amersham Biosciences). The Southern blots were probed with α-³²P labeled BjNPR1 sequence [29] (link). Similarly, northern blot analysis was carried out using total RNA isolated from the untransformed plants as well as transformants. About 10 μg of RNA was separated on 1.4% denaturing agarose gel and α-³²P labeled BjNPR1 sequence was used as probe.

Total RNA was obtained from rice plants as described above. Northern blot analysis was performed as previously described [50 (link)]. RNA (8 μg per lane) was separated by electrophoresis on 1.2% (w/v) formaldehyde-denaturing agarose gels and blotted onto N+ nylon membrane (Amersham; Buckinghamshire, UK). The probes were amplified from PCR products with primers listed in S5 Table, and labeled using α-³²P-dCTP with the Random Primer DNA Labeling Kit (TaKaRa; Shiga, Japan). Hybridization and detection were performed according to instructions that came with the PerfectHybTm Plus Hybridization Buffer (Sigma).