The protocol described here is an adaptation from this paper:Sun, Z. Z., Hayes, C. A., Shin, J., Caschera, F., Murray, R. M., Noireaux, V. Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology. J. Vis. Exp. (79), e50762, doi:10.3791/50762 (2013).Please note that some steps were taken literally as they appear in that source, and It is highly recommended to see and study this paper before starting. Our adaptations include changes in the volumes of cell cultures and buffers, for smaller scale production. Also, it has been eliminated the clarification, ultra-centrifugation, and dialysis steps. Another change is the use of E.coli strain BL21 (DE3) STAR, instead of Rosetta 2 cells. All the above modifications allow the preparation of a simpler, and yet very effective cell extract suitable for cell-free RNAPT7 Tx-Tl reactions.