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Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

Article DOI: 10.3791/62728-v • 07:35 min • June 14th, 2021
June 14th, 2021 •
1, 1
1Chemical and Biological Engineering, Iowa State University

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The study describes a protocol for creating large (µg-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

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Rapid Enzymatic Methods Amplification Minimal Templates Linear Templates Protein Prototyping Cell-free Systems DNA Template Cell-free Reactions Gene Fragment Synthesis Facility Rolling Circle Amplification Traditional Cloning Higher Throughput Design Build Test Learn Cycles Libraries Of Synthetic DNA Inherent Variability Small Volumes PCR Kit PCR Tube Resuspended Gene Fragment Homogenize Mixture Vortexing Thermocycler PCR Cleanup Kit DNA Binding Buffer Spin Column Centrifuge Flow Through DNA Wash Buffer
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