Team:Waseda/Protocol
Protocol
Cell extract(S12) preparation protocol
We based the preparation of E. coli extracts for cell-free reactions on a paper by Jewett [Kwon et.al 2015]. The basic protocol is as follows.
1. Cells were cultured in overnight as pre culture.
2. Diluted overnight cell culture were cultured in fresh medium to OD=3.0 (3~4h)
3. The cells were harvested by centrifuging(7200x g) and washed with S12 Buffer.
4. Cells were sonicated 1 min 50 sec in total( 900 ~ 1200 J)
5. Lysate were centrifuged. (12000 x g)
6. The supernatant after centrifugation was frozen at -80°C ( keep -80 C)
S12 Buffer contained 10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate and 2 mM dithiothreitol. We sonicated cells by sonicator (Sonicator 3000,Qsonia) with microtip(microtip4118, Wakenbtech)
The culture medium and strains used at the time of culture vary from one experiment to another.
Reference
[Kwon et.al 2015] Kwon, Y., & Jewett, M. C. (2015). High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Scientific Reports, 5(1), 8663. doi:10.1038/srep08663