Heparin in PCR?

macklon at acs.ucalgary.ca macklon at acs.ucalgary.ca
Sun Nov 6 17:04:55 EST 1994


In article <393iuv$fhk at owl.csrv.uidaho.edu>, raman913 at uidaho.edu
(Ramanujam Raman) wrote:

> Dear Netters,
> I have been using Heparin as non-specific competitor in my gel-retardation
> assays at the concentration of 10 ug per 15 ul reaction.  I had made up
> a 20 ug/ul stock about 6 months ago and had it stored at 4^C.  I have had
> very good specifc shifts so far. Unfortunately, it isn't working now. In
> the process of trouble shooting the problem, I was wondering about the 
> stability of heparin at 4^C.  Is this storage temperature ideal for heparin
> in solution?  Or does it need to be at freezer temperatures? Also, if anyone
> out there has been using it for gel-retardation assays, is there preference 
> for any particular salt of the compound? I have been using the sodium salt 
> of heparin.  
> 
> Another thing that I would like to know more about is the function of this
> compound.  I know that being a highly polyanionic compound,it mimics DNA
> and binds proteins that non-specifically bind DNA. Further insight into the
> mode of action of this molecule would be very appreciated.
> 
> Thanking in advance,
> 
> RAM.

RAM and fellow netters,

I'd like to add another question regarding heparin: I've heard that it can
be an inhibitor in PCR rxns., which I assume is due to its DNA mimicry.  I
am currently trying to run PCR on HIV from lymphocyte fractions, and the
blood was collected in heparinized tubes. The lymphyocytes have been
washed 2X with TE, treated with SDS and proteinase-K digested, followed by
10 min at 95C. The problem that I'm encountering is sporadic appearance of
the desired band-when it's there it's clean but in low amounts.  I've
tried varying the cell conc., the amount (and source) of Taq, and the
cycling parameters all with the same effect--sometimes it's there and
sometimes it's not, even with the same sample. Does anyone have any
suggestions? Does this sound like heparin inhibition?
(I use a Perkin-Elmer 9600 with thin-walled tubes, so it's probably not a
tube-temperature effect-I think).
May I also add my thanks in advance. (my e-mail is beckthol at acs.ucalgary.ca)
bb



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