urgent help please

I U degrades approximates 1 ug of DNA in 10 min at 37C in appropriate
reaction buffer. Your buffer does not seem to be appropriate since it
has EDTA which is an efficient chelator and will inhibit DNase activity.
It would work well with a sonicator.
Try to use a buffer without EDTA but containing other protease
inhibitors (Roche complete protease inhibitor cocktail is a good
alternative (the one without EDTA)). For optimum activity, DNase
required Mg++ in buffer.
Try out this protocol
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ase/Purification/ec_Ecoli_enzyme.html

Best of luck
Jai



-----Original Message-----
From: [Only registered users see links. ]
[mailto:[Only registered users see links. ].indiana.edu] On Behalf Of chiranjit
chowdhury
Sent: Sunday, May 28, 2006 1:26 PM
To: [Only registered users see links. ]
Subject: urgent help please

Hi!!

I am a research scholar of dept of Biotechnology ,IIT
Kharagpur,India.Myexperiment simply involves growing E coli containing
a pPBP fusion
construct,lysing the cell,purify the protein.Now I don't have access to
a
sonicator.So I simply followed the protocol recommended by New England
Biolabs and prepared a lysis buffer containing lysozyme,EDTA,Tris and
NaCl
(pH8).The volume of this buffer is 50 ml.But the main problem I have
faced
is the suspension becomes highly viscous due to chromosomal DNA and it
becomes very hard to be taken out.I know that DNase could solve the
problem
but I don't know how much should I have to add. My DNase I (RNase
free)conc
is 1U/ul.

With worm regards..



chiranjit chowdhury

research scholar

iit kharagpur

India
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in that case, (as a means of emergency) increase the magnesium
concentration to exceed the chelating capacity of EDTA

Wo

And actually, having 10 mM MgCl2 on its own (in the absence of EDTA of
course) will make the DNA quite easy to pellet so that you can just spin
it out instead of shearing it or digesting it. However, I strongly
recommend adding it at the same time as the lysozyme, NOT after the cells
are already lysed. Granted, it makes it harder to tell when the cells
have lysed, but it works really well for me.
--
__________________________________________________ ____________________________
Lou Hom >K'93
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