Ready to go random primer dna labelling kit

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.

Genomic DNA was isolated from the putative transformants and untransformed control (UC) plants^{62 (link)}. For PCR analysis, primers 5-′GAATTCGAGTTCGCCAGGAACCAG-3′ & 5′-GGATCCGATGATGCTCACGGAACTG-3′ for cry1Ac gene, and 5′-GGATCCGCTATTCTAACCATACTG-3′ & 5′-GAGCTCACCCACA CTTCTTCTGTAGG-3′ for asal gene, 5′-CTACCATGAGCCCAGAACG-3′ & 5′-TCAG ATCTCGGTGACGGG-3′ for bar gene, and 5′GCTCAACACATGAGCGAAAC-3′ polyA reverse primer were used. Later, the PCR products were separated on 1% agarose gel and analyzed.
For Southern blot analysis, 15 g of genomic DNA samples from transformants as well as UC plants were digested separately with Hind III and EcoRI enzymes. The digested DNAs were electrophoresed on a 0.8% agarose gel and subsequently transferred to N+ Nylon membranes (Amersham Biosciences), and were fixed by exposing to UV (1200 J for 60 s) in an UV cross-linker. The membranes were separately probed with cry1Ac::asal and bar coding sequences labeled with α-³²P dCTP, employing ready-to-go random primer DNA labelling kit (Amersham Biosciences). Further, the processing of membranes was done according to the manufacturer’s instructions.