PCR.PROB

mic268a at vaxc.cc.monash.edu.au mic268a at vaxc.cc.monash.edu.au
Sat Jul 25 23:17:09 EST 1992


>I have a colleague who is trying to PCR single
> copy genes from eukaryotic DNA by both normal
> and inverse PCR procedures. 30 cycles usually
> results in faint or non-existent bands being
>present at the appropriate sizes. However, when
> these bands are excised and re-PCR'd, then
>numerous smaller fragments are generated, half
>the size and smaller than the original band. Does
>anyone know why, and/or what can be done to prevent it.

>Thanks in advance,

>Mike Poidinger
>Dept of Microbiology
>University of Reading


Mike,
I have also been attempting similar experiments to your colleague.
I am using inverse PCR to amplify a flanking region to a transposon insertion
Initially I could only see a faint band of the expected size. When I re-PCRed
the PCR fragment I predominantly got a very small fragment and I did not get
the expected fragment.
What I then did was I increased the volume of the ligation from 20ul to 500ul
then I EtoH precipitated the ligation mixture and did the PCR on that.
To my surprise I got the expected fragment and got buckets of the stuff.
My suggestion to your colleague is to increase the volume of the ligation. Your
conditions are probably right, I would certainly not increase the number of
cycles. Hope this is of some help

Mike L.
Monash University
Clayton, Victoria
AUSTRALIA



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