Rediprime 2 random primer labeling kit

KpnI and HindIII cleaved BAC16 DNA was separated on 1% agarose gel and visualized by ethidium bromide staining. The DNA in the gel was transferred to NC membrane using downward alkaline transfer. A K-bZIP probe was radiolabeled with [α-³²P] dCTP (Perkin Elmer) using Rediprime II random primer labeling kit (GE Healthcare, UK). DNA blots were hybridized overnight at 65°C with rotation. After washing, the blots were imaged using X-ray film (Kodak, Rochester, NY, USA).

Genomic DNA was isolated from LB grown log phase cultures (OD600 ~0.3) using the Genelute Bacterial Genomic DNA kit (Sigma). The DNA was digested for an hour with 3 units of MboII or HphI at 37°C, or TaqI at 65°C (New England Biolabs). Partial digestion of genomic DNA from dam minus strains was performed with 3 units of TaqI at 55°C for 10 min. The digested products were resolved in a 1.3% agarose gel. An oriC region (3925517–3925542) was PCR amplified using primers jj40+jj41 and the product was used as the probe. The probe for the HphI digested blot covered the region 3925275–3925633 and was amplified using primers jj168+ jj169. The probe for the oriC external-marker in lacZ covered the region 364871–365085 and was amplified using primers jj193+jj194. The probes were made radioactive using the Redi-PrimeII random primer labeling kit (GE Healthcare) and [α-32P] dCTP (Perkin Elmer). The band intensities were recorded and quantified as described earlier [27 (link)]. The blots were re-probed for an external marker (in lacZ) located ~365 kb away from oriC. As reported earlier, the level of HM DNA at lacZ was significantly lower compared to the levels at TaqI sites created in oriC (Fig 2B).