Hi,
Sadly, that happens sometimes.
1) make sure you have some salt in the DNA stock too
2) try (NH4)2SO4 instead of NaCl (just don't add Ca++, and
remember the ionic strength of a given molarity will be higher)
3) if you have to, try more salt
3) try different ends on your oligos - sometimes they behave
differently
4) examine the "precipitate" under a microscope. Sometimes
it turns out to be microcrystalline.
5) if all else fails, change your fickle-gods-propitiating
protocol
good luck!
Phoebe
---- Original message ----
>Date: Wed, 1 Oct 2008 23:52:32 +0100
>From: E rajakumar <[log in to unmask]>
>Subject: [ccp4bb] Protein-DNA complex prepartion for
crystallization
>To: [log in to unmask]
>
>Dear All
>Sorry for non-crystallography query. I am working on
>DNA binding protein, while mixing DNA with protein for
>preparing Protein-DNA complex for crystallization,
>protein is precipitating. pI of the protein is 9.3
>and in 15 mM HEPES 7.0, 150mM NaCl and 5% glycerol.
>Concentration of the protein used for mixing with DNA
>is 8 mg/mL. DNA to protein molar ratio is 1.2. Please
>advise me how to prevent precipitation. Is changing of
>buffer pH and adding divalent cation like MgCl2 can
>help in preventing precipitation?
>Thanking You
>Rajakumara
>
>
>E. Rajakumara
>Postdoctoral Fellow
> Strcutural Biology Program
> Memorial Sloan-Kettering Cancer Center
> New York-10021
> NY
> 001 212 639 7986 (Lab)
> 001 917 674 6266 (Mobile)
>
>
>
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Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
RNA is really nifty
DNA is over fifty
We have put them
both in one book
Please do take a
really good look
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