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