c was kept for 7 more minutes the pcr products were analyzed in a 1 4 agarose gel and visualized in an ethidium bromide staining under uv light the lowest densely visible bands in each of the five species were excised from the agarose gels and purified using qia quick gel extraction kit qiagen the purified dna was ligated in the pmos blue t vector overnight at 16 u00b0 c as per manufacturers instruction amersham life sciences the ligated mixture was transformed into e coli mos blue cells and selected onto x gal iptg and ampicillin 100 u03bcg ml containing luria bertani lb agar plates ten white colonies identified by colour selection were grown in 5 ml lb liquid medium overnight the mini preparations of the plasmids were performed using alkaline lysis method sambrook j fritsch e f and maniatis t 1989 molecular cloning a laboratory manual 2 nd edn cold spring harbour laboratory press ny the correct dna inserts were verified by restriction analysis finally one plasmid clone from each plant was selected and its dna prepared using qiaquick plasmid dna isolation kit qiagen which was then subjected to sequencing in both the directions using the t7 promoter primers seq id no 11 5 u2032 taatacgactcactataggg 3 u2032 and m13 forward primer seq id no 12 5 u2032 cgccagggttttcccagtcacgac 3 u2032 respectively on an applied biosystems.