poor PCR efficiency

Here we discuss correction for PCR efficiency

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poor PCR efficiency

Postby ravuri » Wed Mar 25, 2015 12:16 pm

Hi,

I am getting good r square value around 0.98 but because of low slope value my pcr efficiency is around 150%. i know there is something wrong. Can you please tell me where is going wrong? Can i redesign primers?

Ravuri
ravuri
 
Posts: 18
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Re: poor PCR efficiency

Postby Mikael Kubista » Tue Apr 26, 2016 10:00 am

Something is clearly wrong. A common reason to obtain too high PCR efficiency is that Cq values of the most concentrated samples are low and the instrument has issued with base line subtraction. Test removing the most concentrated sample from the analysis and redo it. I also recommend doing it in GenEx (can be downloaded from www.tataa.com) to calculate better statistics than r square.

If you need help with assay design, contact us on: services@tataa.com
Mikael Kubista
 
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