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Reamplification mtDNA, should I?

Scrimshaw, Brian J SCRIMSHAWB%GRIEHFS at LINCOLN.CRI.NZ
Tue Jul 12 00:19:49 EST 1994


I am working on some very poor quality DNA samples from bones
(very weathered, but not ancient). I have got a 118bp band from
the hypervariable region I of the D-loop to amplify, after much
effort to extract anything that would amplify and primer
redesign. After 40 cycles and a hot-start I have got a band from
my positive control (strong) plus a very weak band in my
important bone sample lane, plus no amplification from my blank
control. So I'm a very happy bloke. I want to sequence this weak
band, but feel it is too weak to allow any decent recovery. What
I propose to do is to reamplify the weak band to get enough to
sequence. I guess I'll have to also include a new blank control
and reamplify the old blank. After 40 cycles and no
contamination, I'm scared some will sneak in to this
reamplification, What do others think about this approach? In my
opinion it's the best option I have.

ie, if you were reviewing a paper where someone did this would
you mind? (provided all the appropriate controls are run)

Brian Scrimshaw
Wellington
New Zealand



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