Pulsed-field gel and Southern blot analysis of the β3 gene contained in clone D12. Two blots, generated using different electrophoretic conditions, are shown for each of the three probes. Panels are presented according to the 5′ to 3′ orientation of the probes (positions shown in Fig 3A). In all panels, the arrow indicates the 30.5-kb Xho I fragment. The same filter was probed, stripped, and reprobed in (A), (C), and (F ). Size markers are λ DNA PFGE Markers (Pharmacia) in (A), (C), and (F ); and 8- to 48-kb (mixed digest of λ) markers (Bio-Rad) in (B), (D), (E), and (G). (A and B) Southern blot analysis with oligonucleotide probe ex0.S, which is 5′ of the first exon. D12 DNA was digested with the indicated restriction enzymes. The Pst I-Pst I fragment (from the Xho I + Pst I digest) containing probe ex0.S is 1.49 kb and has run off the gel. (C, D, and E) Southern blot analysis with probe int0 between the first and second exons. The ethidium bromide-stained gel in (E) was used for the Southern analysis shown in (D) and is shown to demonstrate the ability to resolve the high molecular weight fragments in these studies. D12 DNA was digested with the indicated restriction enzymes. Note that the Kpn I digested completely in (C), but partially in (D) and (E). In (D) and (E), the ”Marker“ in lane 1 contains 8- to 48-kb (mixed digest of λ) markers (Bio-Rad); in (E), lane 0 contains λ DNA digested with HindIII.
(F and G) Southern blot analysis with probe ex2.A, which is 3′ of exon 2. D12 DNA was digested with the indicated restriction enzymes. The Kpn I-Kpn I fragment (from the Xho I + Kpn I digest) containing probe ex2.A is less than 2 kb and ran off the gel.
