PCR problems

Robert Coelen robert at arbo.microbiol.uwa.oz.au
Mon Jul 20 20:32:20 EST 1992


In article <1992Jul20.122104.5791 at gserv1.dl.ac.uk> skspoidn at uk.ac.rdg.susssys1 (Mike Poidinger) writes:
>I have a colleague who is trying to PCR single
  <deleted stuff>
>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
>
>"All opinions stated are someone elses.
> I don't have any of my own"


Mike, 

How much of the excised band is entered into the second PCR ?

All too often second PCRs fail because of the addition of too much template.
Smearing is often seen, which disappears if the amount entered into the 
second round is reduced (say by a factor of 100 - 1000)

May be Taq is capable of copy choice at low temperatures (when you set up
the second reaction) and if many template molecules are present it 'jumps'
around from strand to strand thereby creating a set of smaller molecules.
Whilst this process could lead to larger molecules initially, I think the
amplification process favours the generation of smaller molecules.

Hope it helps


Robert Coelen
Department of Microbiology, UWA

PS You might like to try something really crazy, but something analogous
worked in our RT-PCR (described in NAR issue 7, 1992): add some unrelated



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