Genomic DNA was isolated from B cells, either resting or cultured with LPS for 4 d. Cell pellets were incubated with proteinase K (0.5 mg/ml), RNaseA (100 μg/ml), and SDS (0.5%) in STE (0.1 M NaCl, 20 mM Tris, 1 mM EDTA) for 2 h at 37°C, followed by 3–4 extractions with phenol/chloroform (1:1) and precipitation with 0.3 M sodium acetate, pH 7, and ethanol. DNA was wound out on glass rods and resuspended in TE, pH 8. The germline Sγ3 segment was amplified by PCR from resting purified B cells from WT(129 × B6) mice for comparison to Sμ-Sγ3 junctions from cells induced to switch to IgG3. Expand HiFidelity Taq polymerase (Roche Laboratories) was used with the following primers: g3–1 (5′CAGGCTAAGATGGATG- CTACAGGG-3′) (MUSIGHANA 404–427) and g3–2 (5′TAC- CCTGACCCAGGAGCTGCATAAC-3′) (MUSIGHANA 2603–2628) to amplify the 2.22-kb fragment of germline Sγ3. Sμ-Sγ3 junctions were amplified by PCR using Expand Long Template Taq polymerase (Roche Laboratories) and the primers μ3-H3 (5′AACAAGCTTGGCTTAACCGAGATGAGCC-3′) and g3–2 (above). The germline Sμ sequence was deduced by comparing the sequences of a large number of Sμ-Sγ3 junctions from wild-type mice. For the sequence analyses, the wild-type sequences from the corresponding littermates were used.