Nytran spc membrane

PCR primers 5BBQ67 and 3BBQ67 were designed to amplify an internal fragment from the bbq67 gene of the B31 genome present on lp56 (Table 1). N40 strain lacks lp56 [27 (link), 50 (link)]. This PCR was performed to test whether N40 genome possesses a homolog of bbq67 elsewhere on the genome. Genomic DNA of B31 5A18 NP1 (genotype lp56^-, lp28-4^-, bbe02::Kan^r) was included in this assay as a negative control, because this B31 derivative lacks the plasmid lp56 [32 (link)]. Genomic DNA isolated from B31 5A4, B31 5A18 NP1, and N40 cultures were digested with restriction enzyme EcoRI. DNA fragments were resolved by agarose gel electrophoresis, and transferred to Nytran SPC membrane (GE Healthcare Life Sciences, PA). Southern hybridization was carried out following the standard protocol to confirm the absence of bbq67-homolog in N40 genome. PCR product amplified from B31 5A4 genomic DNA by 5BBQ67 and 3BBQ67 primers was labeled with DIG using DIG High Prime DNA Labeling and Detection kit (Roche, IN). Hybridization and detection procedures were carried out according to manufacturer’s instructions.