need help: PCR band of right size, but sequencing reveals wrong thing!
junk at hgmp.mrc.ac.uk
Mon Oct 28 07:21:43 EST 2002
Historians believe that in newspost <B9E147C0.8E11%mlevin77 at attbi.com>
on Sun, 27 Oct 2002, Mike Levin <mlevin77 at attbi.com> penned the
following literary masterpiece:
>Now, I understand that degenerate PCR often comes up with the wrong thing,
>but what I can't figure out is how the band size could be correct! If it
>happens to match some random sequence, shouldn't the size be random and
>different too? Plus, this same thing happened with different primers and
>different template choices. So, I suspect that the PCR is working ok, but
>that we have some sort of problem after the PCR step. I'm baffled as to what
>it could be though.
Possibly just pure bad luck that the PCR product has the size you expect
but is the wrong sequence.
What happens when you RE digest the PCR product? Is it a distinct single
product and does it agree with the RE map of your sequenced product. If
so they are obviously the same.
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
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