Amersham rediprime 2 random prime labelling system

For the D. bruxellensis transformants 10 μg of their total DNA was digested with restriction enzymes (either EcoRI or BglII) and then separated on 0.8% agarose gel.
For the strains of D. bruxellensis, their chromosome-sized DNA was prepared (following [39 (link)]), after which their chromosomes were separated by pulse-field gel electrophoresis (PFGE) using a CHEF Mapper XA (Bio-Rad) and the following 4-step programme: (1) a 2700s pulse for 25 h, at 1.5 V/cm and the angle of 53°; (2) a 2200s pulse for 25 h, at 1.5 V/cm and the angle of 60°; (3) a 1500s pulse for 30 h, at 2 V/cm and the angle of 60°; (4) a 500s pulse for 30 h, at 2.5 V and the angle of 60°.
The separated digested DNA or chromosomes were transferred onto the Hybond XL-membrane and hybridized with radioactively labeled DNA probes. The DNA labelling was performed with [γ-³²P] dCTP using the Amersham Rediprime II Random Prime labelling system (GE Healthcare, Freiburg, Germany). The signals were detected using the Imaging Screen-K (35×43 cm; Bio-Rad) and Personal Molecular Imager FX (Bio-Rad).

Genomic DNA (10 ug) cleaved with SacI was transferred to Amersham Hybond-N⁺ membrane (GE Healthcare, Chicago, IL, USA) by 10 × SSC using Model 785 Vacuum Blotter (Bio-Rad, Hercules, CA, USA). [α-³²P] dCTP-labeled hpt, gus, gfp, and bar fragments with Amersham Rediprime II Random Prime Labelling System (GE Healthcare, Buckinghamshire, UK) were used as hybridization probes. Hybridization and washing methods were according to Sambrook et al. [38 ]. After washing, the membranes were exposed to a phosphor screen for 5–12 h and scanned on Typhoon FLA 9500 (IP: 635 nm, PMT: 500 V, Pixel size 200 μm). Probe primer sequences are presented in Table S2.