PCR reamplification problem

BIOCHEMISTRY klautky at vaxa.weeg.uiowa.edu
Sun Jul 7 04:10:42 EST 1991

Dear Net Readers,

    I am posting this request for a fellow colleague -> Lando Torres.

    PCR amplification problem:
    A 700bp fragment previously synthesized by PCR does not amplify
properly when diluted and amplified with the same primers.  Is there a
common reason for this sort of problem or is this very unusual?  I
wanted to use the synthesized fragment as a stock to make more when I
need it for probing, sequencing etc.

    This fragment was made after a reverse transcription reaction of
RNA using a third primer.  The result was run on a SeaPlaque gel and
the band of correctly predicted size cut out.  There were no other bands
on the gel but there was some background above and below the band (band
was distinct).

    The fragment was then isolated by excision and purified by
freeze-fracturing the gel, phenol/CHCl3, EtOH ppt.  When submitted to
the same reaction conditions (except the reverse trans.) as before the
result on the gel is bizarre.  1 ng of starting material (30 cycles/100ul
rxn. all expts.) gives no distinct bands instead a smear starting at the
well and varying in intensity on down to where the primers are is present.
There appears to be a very broad "band" (more like a smudge) where the
expected product is suppossed to be.  At more dilute starting amounts
(100 pg and 1 pg) amplification also occurs but there are still no
distinct bands only a very broad band near the expected product location
with a smear below the "band" but no smear above.

    Initially, it was suspected that an electrophoretic problem was the
cause so the sample was diluted and also run on a lower and higher
percentage gel - same result.  The PCR buffers were also checked for
contamination and both primers and substrate are necessary for the
abberrant amplification.

    Any ideas would be helpful at this point.  Thanks.

    Lando Torres


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