Comparative Physiology @ Guelph
New WSU TnI Pellet Protocol
Use freshly transformed BL21 cells!
- Mid-afternoon, 4 L of LB + 30mg/L Kamamycin inoculated with 20
- Â Â Â Â Â Â Â Â Â Colonies in each 2L flask, then grow overnight 37oshaking
- Take 1 ml sample of O/N growth for SDS Page analysis
- Spin culture at 7,000rpm for 5 minutes to pellet, toss super
- Resuspend pellets in 200ml Sonication Buffer
- Sonicate 10-15 times on high power for about 1 minute-don’t foam
- Let solution stay on ice while sonicating, let rest between bursts to cool
- When finished, solution has consistency of water-test with pipet bulb
- Rinse probe with dd water, EtOH, dd water again
- Spin 10,000rpm 1 hour Save super for column purification
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Sonication Buffer-pH 8 at 4o       1 liter
6M Urea                                   360.36 g urea
50mM Tris pH8Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 50 ml 1 M Tris
5mM EDTAÂ Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 10 ml 0.5M EDTA
0.4mM PMSFÂ Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 4 ml 0.1M
cocktail optional due to 6M urea