Figure 2. Method and primer design optimisation.

(a) Performing PCR with no homologous regions highlights potential false positives arising from template DNA. Increasing template DNA increases the number of colonies produced on transformation (■ purple), independent of PCR amplification (● magenta). 1 ng regularly produces 0 colonies, yet gives substantial PCR amplification (dashed line). (b) Relationship between increasing length of homologous regions (constant T_m) and colony yield shows little length dependence of recombination above 15 bp. (c) Increasing the T_m of homologous regions increases the colony yield and hence recombination efficiency. (d) Bar chart indicates that IVA cloning colony yield (purple) is reliant on amount of PCR product (magenta) independent of the type of PCR polymerase. (e) Properties of optimum primer design to maximise recombination efficiency. Homologous regions are included in 5′-end of primers, homologous to a region (orange) of the partner primer. Template binding regions are shown in green.