Figure 1.

Summary of conditions tested for the preparation of mycoplasma lysate and its functional assessment. The lysate preparation starts with harvesting mycoplasma cells (upper left). For some preparations, cells were trypsinized before cell disruption. To obtain crude lysates, we initially tested different cell lysis methods: sonication, French press, osmotic shock and liquid nitrogen grinding (lower left). In the first centrifugation step, most of the cell debris are decanted, and the supernatant (clarified lysate) proceeds to run-off incubation, second centrifugation, dialysis (in S30B buffer) and third centrifugation. The final lysate is then flash-frozen and stored at −80°C (lower right). To test lysate functionality, we performed CFE reactions and measured transcription and translation by tracking fluorescent probes. Some lysate preparations included cell trypsinization before lysis. *The second and third centrifugation speeds are the same as the first centrifugation step.