my pcr bands are at the required size compared with ladder. and there are no bands in negative control. additionally, i cultured Dh5alpha ecoli cells for one day and used for transformation when od was 0.4 . further i am using kanamycin as selection marker and colonies were observed after 14 hrs incubation(acc to the pET manual, kanamycin shows no satellite colonies this early). I also plated my e coli cells in kanamycin plate and observed no growth. but i can't extract my plasmid after ligation and transformation. The gel shows no band.