# TX-TL Crude Cell Extract Prep

`PROTOCOL DUMP PLEASE FORMAT`

## CCE Prep Protocol – Fermentation Facility

1. Prep 20 L 2xYT + P + 40 ug/mL chloramphenicol.
2. StreakE. coliBL21 Rosetta 2 on 2xYT + P + 40 ug/mL 1.5% agar plate. Growovernight.
3. Inoculate 1 colony into 300 mL 2xYT+P+40ug/mL to make seed flask. Grow tomid-log.
4. Inoculate 20 L 2xYT+P+40ug/mL with seed flask. Grow \~3 hrs, until OD = 2.0 \[Note: I’m 90% sure this equates to mid-log growth in this media]
5. Collect cell pellet with continuous flow centrifuge.
6. Add 6 mL cold S30A buffer per g cell pellet. Use overhead mixer to resuspend.
7. Spin down 15 min, 7000 g, 4C. Decant supernatant.
8. Repeat wash step.
9. Resuspend cells in 1 mL cold S30A per g cell pellet.
10. Run cells through microfluidizer 2x. (cell suspension will change from beige to brown – get darker – as cells are lysed)
11. Centrifuge lysate at 14k RPM (\~14k g) for 1 hr at 4C.
12. Siphon off supernatant and incubate at 37C, shaking, open to air, for 80 min.
13. Spin down 1 hr, 14k RPM, 4C.
14. Filter thru 0.1 um filter to remove any remaining large cell debris/cells.
15. Diafiltration on filtrate for 22 hrs, using S30B buffer. Run 2-3 retentate volumesthrough. Do not concentrate!
16. Measure protein concentration (should be 20-30 mg/mL).
17. 1. In the final TX-TL setup, CCE is 1/3rd of reaction mixture, leading to afinal protein concentration of 6.7-10 mg/mL


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