Biotin Pcr primers and anealing temperature

Karl Voss karl at hobbes.chem.ualberta.ca
Fri Aug 23 12:29:35 EST 1996


John Dixon (jpcd0 at mole.bio.cam.ac.uk) wrote:
> In article <4vi08d$blo at pulp.ucs.ualberta.ca>, karl at hobbes.chem.ualberta.ca
> (Karl Voss) wrote:

> > Hi,
> > 
> > Does anyone know if having 5' biotin on pcr primers lowers the anealing 
> > temperature that they need.  I have been getting very good pcr results with
> > a non-biotinilated primer set that have short 5' sequence overhangs to be
> > used for directional cloning.  I wanted to directly sequence the pcrs so 
> > I got a biotinilated primer made that did not have the extra 5' bases.
> > The resulting pcr bands are very weak.  I have quantified the primers 
> > carefully and assume that they are of high purity (they were purchased from 
> > Research Genetics).
> > 
> > Has anyone run into similar situations?  If so I would like to hear
> > from you.
> > 

> Hi Karl, I have just made a biotinylated primer that exactly matches a
> primer made for somebody in the lab 5 years ago that still works fine. The
> new biotinylated one completely fails, even on pure plasmid, while the old
> one PCRs from the genome (mouse) no worries. I am in the process of
> complaining to the oligo service but the spec analysis looked fine. I have
> to say that I have only tested the new one uneder the exact conditions
> used for the old, so that if the annealing temperature has changed
> dramatically, this might explain it some of the problem.

I just tried out the new primer at three degrees lower anealing temperature.
Works OK.  Looks like the biotin may interfere with primer template 
hybridization.  PCR seems to depend on the phase of the moon as much as
anything else and I guess the rule is that if you change anything all the 
conditions that you worked out don't count no more.  

Good luck.

Karl

--
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If it ain't broke it doesn't have enough
features yet

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Karl Voss
Department of Chemistry
University of Alberta
Edmonton, Alberta, Canada
T6G 2G2

karl.voss at ualberta.ca
phone 403-492-0222
fax   403-492-8231



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