Zero blunt topo cloning system

For Sμ-Sα junctional analysis, 100 ng of genomic DNA isolated from CH12 stimulated with CIT for 72 hours was PCR amplified with primers described in supplementary table 2 using the Phusion polymerase system (Invitrogen). The PCR conditions were 98 C for 3 minutes; 98C 30 seconds, 60C 30 seconds and 72C for 2 minutes for a total of 38 cycles. The PCR products 1–2 kilobases long were gel purified and cloned using the Zero blunt topo cloning system (Invitrogen). For Sμ-Sγ1 junctional analysis, 100 ng of genomic DNA isolated from Hmces^+/+ and Hmces^−/− primary B cells stimulated with anti-CD40 and IL-4 for 4 days was PCR amplified with primers described supplementary table 2 using the Phusion polymerase system (Invitrogen). The PCR conditions were 98 C for 3 minutes; 98C 30 seconds, 58C 30 seconds and 72C for 2 minutes for a total of 35 cycles. The PCR products were purified and cloned using Zero blunt topo cloning system (Invitrogen). DNA from individual bacterial colonies were sequenced using Sanger sequencing (MCLAB) and aligned to switch-mu (MUSIGCD07), switch-alpha sequence (MUSIALPHA) and switch gamma 1 (MUSIGHANB) using Mac Vector software.