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Nested PCR

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2 replies to this topic

#1 molmicro



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Posted 02 September 2003 - 11:45 PM

Has anyone experience of nested PCR? Is it normal to have 2 bands present in the 2nd round of PCR. One band is from the original PCR and the 2nd is from the 2nd round. I am assuming that there is carry over from the first PCR - is this normal? I have tried reducing the amount of PCR product used as template for the 2nd round of PCR however that doesnt seem to make a difference.

#2 hula



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Posted 03 September 2003 - 08:26 AM

How much of your first pcr product do you use? You can dilute your first pcr raction 1:50 or 1:100, then there will be no band from the first pcr show up.


#3 deepee



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Posted 24 September 2003 - 04:13 PM

You think that's bad! I get products of the 1st round and 2nd round, AND cross amplification between the 1st and 2nd round primers, so I usually get 4 bands! Without adding an extra cleanup step (via exo/SAP or a spin kit etc.), which of course adds to the risk of contamination, we've come to the conclusion that it's just an artifact of nested PCR's. It can be reduced by reducing the concentration of the primers in the first step or as the last person said, reducing the amount of 1st round reaction you carry over to the 2nd, BUT this will reduce the sensitivity of the whole process and you may not pick up the band you want, particularly if the copy number in the original template is low, as is the case with us.
If you do figure out a way to deal with it you're better than us, please let me know.

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