incredible PCR results, who can help explain?

Brett Lindenbach brett at BORCIM.WUSTL.EDU
Mon Dec 14 11:54:09 EST 1998


Dear Stupernova,

	If indeed your primers can anneal except for the terminal A, perhaps
you should consider the fact that oligos are seldom free from incomplete
products, unless you specifically purify them based on size, and even then
you might have some misincorporations.  Hope this helps.



>Who has met the problem that a PCR reaction without any template can also
>generate some incredible products? Our product is a series of primer repeats.
>Our primer is a 30-mer designed by Primer Premier. And the both ends of the
>primer are ìAîs. What ridiculous is that the product is a head-to-tail
>repeats. It deletes one base of ìAî at the head/tail and then ligate the
>remaining, like the generation of a peptide bondñdelete something and then
>ligate. Our PCR reaction is performed in a Clontechís Polymerase Mix system.
>
>The PCR profile is:
>94 Centigrade x 0.5min for denaturing;
>then touchdown at 72C and 70C respectively of 5 cycles, 2.5min for each cycle;
>finally at 68C for 30 cycles.
>
>I think the primers canít form a dimer (if this kind could be regarded as a
>dimer) at the temperature around 70C. Furthermore, itís not base
>complementation here, neither annealing. And only knockout ONE base each time.
>Itís incredible and unexplainable. The product is confirmed by sequencing.
>
>Who can help me then?
>
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