Figure 4.

Overlap PCR to generate membrane anchored IgM configuration of germline reverted antibodies. Two fragments, the variable region (+leader sequence, underlined) and mIgM constant region are first amplified by individual PCR. Primers for these reactions are displayed in color. We recommend a minimum of 20bp overlap for the design of primers 3 and 4. Following amplification and gel purification of the two fragments, they are combined and subject to another round of PCR using only primers 1 and 4. The resultant combined fragment should run at ~1.8 kb. Primers 1 and 4 should be designed with the appropriate restriction enzyme sites to ligate the combined product into the desired expression vector.