This collection contains the protocols and recipes for the preparation of cell-free RNAPT7 transcription/translation coupled reactions. The crude extracts are obtained from IPTG-treated E.coli BL21 DE3 STAR cells. IPTGtriggers RNAPT7 expression and accumulation in cells before collection.Reactions take place when the cell extract is mixed with a maltodextrin-based energy solution along with amino acids solution and other salts (Magnesium and Potassium). This system allowstranscription/translation coupled reactions from plasmid DNA that contains T7 promoter transcriptional units. In our hands, ithas been possible to express different fluorescent reporters, including deGFP which is highly efficient. Products of the reaction can be measured during the whole reaction time in a plate reader with appropriate optic filters, open hardware cameras, andmicroscopes. When set up correctlly, deGFP fluorescence intensity is detected as fast as ∼30 minutes afterthe reaction starts and it shows a rapid increase during the first ∼2.5-3.0 hours. The system presented here contains the features highlightedbellow,and is an adaptation of protocols and findings from the following listed papers:1.- This system uses maltodextrinand Polyphosphate-based energy solution: Kim, H.-C., Kim, T.-W., Kim, D.-M., . Prolonged production of proteins in a cellfree protein synthesis system using polymeric carbohydrates (2011) Caschera, F.; Noireaux, V., A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system. Metabolic engineering (2015) 2.- The S12 crude extract is prepared from E.coli cells ( expressing Pol T7 ) using bead beater. 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. Journal of visualized experiments (2013) Kim, TW., Kim, HC., Oh, IS. et al. A highly efficient and economical cell-free protein synthesis system using the S12 extract of Escherichia coli. Biotechnol Bioproc E (2008) 3.- The amino acidstock solution contains each amino acid at ∼12 nM in a stable solution: Caschera, V. Noireaux Preparation of amino acid mixtures for cell-free expression systems. Benchmarks (2015)