Fig. 2.

Fab HC library construction and characterization. (a) The template plasmid containing the trastuzumab HC Fab is used to generate four separate PCR fragments using four sets of primers. The residues in CDR H1, CDR H2 and CDR H3 are randomized using degenerate base pairs (stars) in the H1-H3 primer mixes. The final library is constructed by overlapping PCR using these individual fragments. (b) The final crude assembled library consists of a single band of the desired size (1103 bps) on an agarose gel. (c) Pooled sequencing of the final naïve library shows the intended degeneracy in the coding region for CDR3. See Supplemental Fig. S2 for pooled sequencing of the coding regions of CDR1 and CDR2.