poly dI-dC versus salmon sperm DNA

Byung-Hoon Kim byung-hoon.kim at uni-tuebingen.de
Thu Dec 23 05:08:50 EST 1999



Eric Chace Olivares schrieb:

> Sybille Rex (srex at bu.edu) wrote:
> : Hello everybody,
> :
> : does one of you know if I could use salmon sperm DNA instead of poly
> : dI-dC in a gel shift assay (electrophoretic mobility shift assay) to
> : reduce non-specific binding?
> : Which company is selling poly dI-dC?
>
> Hey Sybille...
>
> I've just begun doing such experiments, and I've been trying to answer the
> same question.  While I am using E.coli crude extracts and purified
> proteins and experience very little non specific binding, I'll tell you
> what I've thought about the subject and maybe we can come to a collective
> decision.  Keep in mind that these are only my misguided opinions...so
> please correct me if I say anything out of the ordinary.
>
> Salmon sperm DNA is a natural competitor, so for any given DNA binding
> protein, it may or may not contain binding sites that can "soak up" the
> non specific proteins in your preparation.  Poly(dI-dC) is a fairly
> regular synthetic heteropolymer that has been shown to help in reducing
> non-specific binding by being a general substrate for DNA binding
> proteins.
>
> I guess what I'm trying to say is that ssDNA might work depending on what
> protein is responsible for the non-specific binding and the strength of
> the interaction.  I have had success eliminating some ghostlike bands by
> using ssDNA in the binding reactions (0.1-10 ug per 20ul reaction)
>
> Another point that I've come across is that it isn't really the content of
> the non specific competitor DNA, but the quantity present.  If your probe
> is the only DNA present, any DNA binding protein will tend to bind it.
> However, if you add cold DNA that doesn't contain your binding site, your
> protein will remain bound to your probe, while the other bunch of DNA
> binding proteins will be spread out over the entire DNA content of the
> reaction, reducing the non specific retardation of your probe.
>
> In summary, I've had some luck with ssDNA, it's cheap (you probably have
> it already), and these experiments are SO easy to run that there is no
> reason not to perform a pilot with it.  BTW, you can get poly(dI-dC) from
> Amersham-Pharmacia-Biotech.
>
> Good luck...and keep us posted. And let me know if you need anymore
> assistance.
>
> -=Eric
>
> --
> Eric C. Olivares
> Department of Genetics
> Stanford University School of Medicine
> olivares at stanford.edu
> http://www.stanford.edu/~olivares/

Hi,
It's nice to know that there are peoples who are doing the same things which I
plan to do.
In fact, I'm also planning to do my DNA-Protein interaction using salmon sperm
DNA as nonspecific competitor.

My question is :  1)  Doesn't dsDNA work at all?
                          2)  Can a normal plasmid (linierized) for cloning be
used for this purpose?


Byung-Hoon Kim
----------------------------------------
Dept. of Gerneral Genetics
Center for Plant Molecular Biology
Univ.of Tuebingen, Germany
----------------------------------------





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