re-amplication of PCR product without internal primers
Andy Law Big Nose
lawa at bbsrc.ac.uk
Thu Aug 25 04:46:22 EST 1994
In article <01HG3ABNW3QQ8WXHAF at cphkvx.cphk.hk>, BHWFDUNG at CPHKVX.CPHK.HK wrote:
> I need to re-amplify a PCR product, using the orignial primers for PCR
> with the gel purified product as template, because the yield of the
> particular fragment is so low.
> Since I don't have much information about the DNA sequence and I can't
> design the internal primers for re-amplication.
> What I got was always a smear even though I use as little as 0.1ng of my
> purified product as template.
What did your original product look like ( before you reamplified it? ).
Are the primers specific for your sequence (are there a family of gene
products that may be interfering). Are the primers designed in one species
and used in another?
You could tinker around with the conditions for PCR (increasing
temperature is one possibility), but you may be there for ever doing that.
If you have 0.1ng of purified product, I would suggest that you clone the
original product into a T-tailed vector then grow a few colonies up and
either PCR from the colony plasmid DNA (that way you know that your
template is homogeneous) or sequence a few of them. If you sequence them,
you also get the information you need to design internal primers.
( Lawa @ bbsrc.ac.uk Big Nose in Edinburgh )
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