Biotin Pcr primers and anealing temperature
jpcd0 at mole.bio.cam.ac.uk
Thu Aug 22 12:59:09 EST 1996
In article <4vi08d$blo at pulp.ucs.ualberta.ca>, karl at hobbes.chem.ualberta.ca
(Karl Voss) wrote:
> 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.
John Dixon Lab 44 (1223) 334131
Wellcome/CRC Institute Fax 44 (1223) 334134
Department of Genetics
United Kingdom e-m: jpcd0 at mole.bio.cam.ac.uk
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