Southern blot hybridization

Biotinylated probes for Southern blotting were prepared by the Biotin DecaLabel DNA Labeling Kit (Fermentas). Template for the probes was pAL-ID plasmid (containing regions present in expression plasmids p1.1 including origin of replication, a beta-lactamase gene, EMCV IRES, DHFR ORF [17]) or a PCR product corresponding to the fragment of FVIII-BDD ORF. Genomic DNA was digested with EcoRI (Fermentas) for 16 h, precipitated by ethanol, separated by 0.8% agarose gel. Gel transfer to an Amersham Hybond-N+ membrane (GE Healthcare, USA) was performed according to the manufacturer’s protocol in 20x SSC buffer (3 M NaCl, 0.3 M Na₃C₆H₅O₇) for 16 h. DNA was fixed by heating the dried membranes at 80 °C for 2 h. Prehybridization and hybridization were conducted according to [22] in the buffer containing 7% SDS, 0.5 M Na₂PO₄, pH 7.2, 1% BSA for 16 h at 65 °C. Membrane was washed according to the manufacturer’s protocol and stained by Biotin Chromogenic Detection Kit (Fermentas).