Figure 4.

A roadmap of protocols for application of cloning by in vivo assembly. Cloning can proceed through four routes for generation of homologous regions. DNA cleanup is required prior to transformation in each case to remove original template circular DNA. Fragments can be amplified either using a single-tube (1) or separate tube (2) PCR, with template DNA destroyed by DpnI incubation. Unamplifiable vectors can be linearized by restriction digestion (3) and co-transformed with PCR-amplified insert containing homologous regions. Finally, direct co-transformation of synthesized dsDNA with target vector DNA linearized by PCR or restriction digestion can be employed (4).