Assembling Linear DNA Templates for In Vitro Transcription and Translation

Cell-free expression systems provide straightforward access from genes to the corresponding proteins, involving fewer handling steps than in vivo procedures. A quick procedure to assemble a gene of interest into a linear DNA template together with 3′- and 5′-untranslated regions using a coupled uracil-excision–ligation strategy based on USER Enzyme and T4 DNA ligase. This methodology will be useful for repeated cycles of expression and in vitro selection, in which gene libraries are repeatedly assembled and their products and templates regenerated.