DNA Directed in Vitro Protein Synthesis with Escherichia coli S-30 Extracts

A DNA-directed cell-free protein synthesizing system was originally developed by Zubay (1). The system contains a crude extract prepared from Escherichia coli. This extract contains the machinery necessary for the transcription and translation, i.e., ribosomes and RNA polymerase. To this system, it is necessary to add all 20 amino acids, all four ribonucleotide triphosphates, transfer RNA, an energy generating system, and various salts. The DNA template is incubated with this mixture for at least 30 min at 37°C before gene products are examined. The following method is essentially as described by Zubay (1), but with minor modifications as described by Valentin-Hansen et al. (2).