Help with sonication of bacteria protocol - my first time! (Feb/07/2007 )

I need to disrupt 200 ml of E. coli BL21 culture.

I found a protocol and it says:
- resuspend the pellet in 10 ml 20 mM tris-HCl, 200 mM NaCl y 1 mM EDTA.
- incubate some hours at –20.
- defrost and sonicate a 15 W by pulses of 20 seconds.

In my new sonicator you can choose the amplitude (0-100%), and you can see W but not choose it.

What amplitude should I use and how many pulses are needed???
How can I know cells are disrupted??????

This is the first time I do it!

Any other protocol or suggestion???

Thanks!!!

---

Hi princess!
A couple of points. First of all, the thawing stage from -20 should have disrupted many of the cells, so the sonication is just to finish off the job. That means, you don't have to bee too harsh in the process, or you could start causing aggregation of proteins (a random process, but one that will seem to affect your protein more... go figure!).

Not knowing which sonicator you're using, I can only give general ideas. I'm guessing that you control to power by altering the amplitude. Start off with low amplitude, check the power output and adjust accordingly. As for knowing when to stop, there are two things that I typically did. Usually, I would look at the tube to see if the solution went from opaque to clear (but coloured, if you know what I mean). Sometimes, however, I would take a drop of the lysate, and look under a microscope for the presence of whole cells. As a ballpark number, I'd do 3 or 4 cycles of sonicating, then check.

Most important of all, however, is to try to keep the system as cool as possible. The sonication process can generate lots of heat, which is why the pulses are limited to ~20 secs. In between pulses, cool the tube in ice or ice-water slurry for about 30 seconds. If yiou have a large volume, you could split it into two tubes, and alternate the sonication and cooling steps.

As for other protocols, you could do two or three cycles of freeze-thawing. You should add lysozyme to assist breaking the cell wall. and DNase I to break the genomic DNA (and protease inhibitors, of course to stop the cells from chewing up your precious protein). Snap-freeze in liquid N2, or the good old standby, dry ice in ethanol (~-80 C), warm to 37C for 10 minutes or so, then repeat. The good thing about freeze-thaw is it is very gentle, and you reduce the risk of aggregating your proteins.

Good luck!

---

THANK YOU VERY MUCH!!!

YOU'VE BEEN VERY HELPFUL TO ME!!!

---