Pfu degrades primer from 5´end??

Georg Wille moqch at mlucom6.urz.uni-halle.de
Fri Nov 13 05:35:44 EST 1998


Geoffrey Kidd wrote:

>Yes, it is possible that your primers have a high content of incompletely
>synthesized primers.  The coupling efficiency is usually 98-99% for each
>reaction in the synthesis of an oligo, if you're dealing with a good
company.
>This means that for a 20-mer, only about 67-82% of the product will be
>full-length.  Since chemical synthesis occurs in the 3'-5' direction, the
>incomplete oligos will be missing the 5' end.  Regarding your observation
that
>different PCR conditions seem to affect the severity of the problem, this
may be
>due to the influence of the various PCR conditions on the ability of the
shorter
>oligos to bind to the template.  Good luck!
>
>Geoffrey Kidd, Ph.D.
>Technical Services Manager
>Aptagen, Inc.
>www.aptagen.com



Though less than 100% coupling efficiency would mean some amount of shorter
oligos, those should not generally be lacking the 5'-end but rather missing
a random nucleotides within the sequence, as the coupling may continue after
a "miss", no?

Georg Wille






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