To sonicate a large volume of bacterial cells - Any better method? (Mar/02/2006 )
I need quite a lot of my bacteria. Every time, I will grow around 15L of cells and do protein extraction by sonication. It is very labor-intensive to sonicate my bacteria in a 10-ml aliquot scale, in which I have around 200ml suspension everytime! Does anybody have a better suggestion for me? I have tried to switch to a larger probe and sonication 100ml each time, but the bacteria seems still alive happily after sonication with the large probe! Please help ~~~
Why don't you treat the cells in your lysis buffer with lyzozyme first ?! In my lab, after centrifuging and resuspending the cells in lysis buffer, we add lyzozyme and incubate at 370C, 30 minutes, then sonicate ... Everything seem so good.
you could try cold lysis (put in lysis buffer and incubate at 4oC) this has worked at comparabe levels to sonication in preparation of extracts from bacillus and pseudomonas
another option is the french press if you have access and enough strength that will sort your bugs out no problem
Can you do a freeze-thaw? Harvest the cells, resuspend in a few hundred ml, add some lysozyme and DNase I, snap-freeze in liquid N2, warm to 37 for ~10 min and repeat. Don't forget the protease inhibitors! Reasonably quick and gentle, and you don't have to be there the whole time!