Figure 2

Phi29 DNA polymerase can be used for highly efficient shRNA template generation. Taq likely encounters difficulty when attempting to read through highly structured templates such as shRNA, despite the inclusion of additives designed to overcome secondary structure. (a) The extension reaction using Phi29 is the only reaction to generate detectable full-length double-stranded product comparable to the annealed complementary oligos. (b) Screening via colony PCR (across the insertion point) reveals that out of all the extensions reactions, the one using phi29 DNA polymerase yields the greatest number of colonies (Total colonies) and recombinant clones (+).