The scale-up of cell-free protein synthesis reactions involves the preparation of large amounts of template DNA. While ion-exchange column chromatography methods have commonly been used to obtain purified plasmid DNA for cell-free protein synthesis reactions, these methods are costly and difficult to expand to a large scale. In this work, we report that the routine isopropyl alcohol (IPA) precipitation method can be used to prepare cell-free-expressible DNA when the co-precipitated proteins are removed. Compared to column-purification procedures, the IPA-precipitation offers obvious advantages with respect to the cost and scaling-up of template preparation, and we believe that our finding will contribute to making cell-free protein synthesis system more practical for the rapid production of preparative amounts of recombinant proteins.