PCR amplifycation of small (`100bp) DNA fragments ?
hubahopp at gmx.de
Tue May 20 09:11:51 EST 2003
Henk and Raffael,
You'll need a careful amplicon design to avoid the PCR product priming
itself and generating smear.
I suppose that PCR conditions are very critical (to exclude formation of
polymers) and you probably want to follow a 2 step scheme just switching
between melting and annealing; enough extension happens already during the
heat up ramp.
At 14:26 20.05.2003 +0200, Rafael Maldonado wrote:
>Henk Bolhuis wrote:
>> Does anyone have experience in PCR amplifying small DNA fragments ?
>> we are trying to amplify a DNA fragment of approx 100 bp between two
>> inverted repeats (a putative bindingsite for a plasmid replication protein)
>> using only 1 oligonicleotide primer, directed against the repeat.
>> We tried several conditions, with Taq or Vent pol. with various amounts of
>> DMSO (up to 10%).
>> Any suggestions ?
>The problem is not the size of the insert, that's for sure. I have made
>PCR of one nucleotide (plus the primers) and they work great. I guess
>that the repeats are affecting the reaction.
>Rafael Maldonado, Ph. D.
>Division of Genetics
>University of Alicante
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