Ultrasonic Homogenizer question....

redeamer alion85 at hotmail.com
Sat Jun 5 18:46:50 EST 2004


Hi OK!

I would recommend you to use such things as LYSOZYME, if you express
in E. coli, or freeze-thaw techniques for disrupting the membranes and
cell walls, etc...

You must shear the DNA --> no Qs about that, Dr Buxbaum is absolutely
right.

But my experience with SONICATION is negative, i mean without lysozyme
it not so good. Because of this "empiricity" you do not often have
reproducible enough results and the protein yield varies.

I would suggest you 1. Lysozyme/freeze-thaw -> 2. sonicate (briefly,
just to SHEAR the DNA, you will understand that => lysate will become
fluid with minimal viscosity, and when you pour it into centrifuge
tube its drops wont stretch) ->
3. then fuge the lyzate some 40 000 g, take super and have fun!

suggest you BATCH! BIND in BATCH! WASH in BATCH! then LOAD COLOMN and
ELUTE (of course all this is applicable if you are doing AFFINITY
CHROMATOGRAPHY[such as Ni-NTA, GST...])

-----> Drew



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