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Really weird amplification curves


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#1 madrius1

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Posted 17 March 2011 - 06:59 AM

Hi guys,

I've suddently had very weird amplification curves in my qPCR. I use 7500 model from ABI. The master mix we use is home made, and was working very well a few days ago. Here's what the curves look like.

First the amplification : ChIP CBP test.sds_AmpPlot.jpg

Then the dissociation curve : ChIP CBP test.sds_DissociationPlot.jpg

And i have a clean band on agarose gel.

I've tried changing the caps ontop of the plate, tried new films, tried another machine, made new master mix, used new Taq, redone my dNTP mix, primer mix. I'm thinking this may be a mix proble, since i have a clean amplification on gel. It maybe the SYBR green, although its seems rather unlikely that both the one in the master mix and the stock solution died at the same time..

Do you have any inputs?? Of course, this is the last experiment asked in review for my paper.. so any quick response would be appreciated. Thanks!

#2 almost a doctor

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Posted 17 March 2011 - 08:35 AM

Seeing how you've got a band in agarose gel, and your melting curves look perfect, all I can think of from your graphs is that the amplification plots are in Rn rather than delta-Rn. Not sure that will solve the problem, but will definitely make your graph look a lot different.

Another option I can think of is that the plate in the qPCR is dirty, but that usually gives nonsense fluoresence all over the place...

Hope this helps.




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