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High efficiency

High efficiency - Real-Time PCR and Quantitative PCR Forum

High efficiency - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 11-17-2009, 12:04 PM
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Question High efficiency



Hi everyone
I am doing qPCR in Corbett machine and having some problems with a really high efficiency. Iíve tried almost everything that Iíve read could cause that problem:
1. Increase the Temperature
2. Change the amount of the MgCl2, which was a slightly better, but still 130-150 % efficiency
3. Change the amount of the primers
4. Played with the dNTPs
5. Make different dilutions

By now I am using only DNA from Promega (female) and this is the only one thing that I didnít change just because it is standard DNA and I donít think it could be the problem. Have anyone had the same problems with the standard DNA? And do you have any ideas what could couse the problem?

Thanks

Katya
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  #2  
Old 11-18-2009, 10:18 AM
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Default Re: High efficiency

High efficiency may be caused by amplification of unspecific products together with specific ones. Check for primer-dimers, specificity of your primers and possible contamination problems.
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Old 11-18-2009, 11:46 AM
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Default Re: High efficiency

Thanks for your replay

I've already check it : the agarose gel and the melting curve are OK, for the specificity according to UCSC the primers are OK too
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