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CT values of qPCR

CT values of qPCR - Molecular Biology Techniques

CT values of qPCR - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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Old 07-03-2013, 05:28 PM
Pipette Filler
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Default CT values of qPCR



Hi everyone,

I知 fairly new to qPCR and I知 not sure how to troubleshoot the problem I have.

I知 running a duplex qPCR for two splice variants of my protein of interest. I知 using Syber since I知 more interested in the melting curve at the end, to screen for the existence of the isoforms. And that actually works. I can detect single peaks at different temperatures for homozygous or double peaks for heterozygous forms. My template is genomic DNA.

The problem is that I have to go up to 45 cycles to do this. As far as I understand qPCR should give a CT value of not to much higher than 25 to be trusted. My CT values are always between 33 and 36. I have increased primer, screened from 10 pmol and 50 pmol, increased template concentration, screened from 35 ng to 140 ng, added various amounts of Mg, tried different reaction volumes, 25 and 50 ul and used various annealing temperatures. Nothing seems to work. I always get the right melting curves but the CT does not change at all. Always 33 36.

Is there anything else I can try? Any suggestion is greatly appreciated.

C
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Old 02-15-2014, 11:37 AM
Pipette Filler
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Default Re: CT values of qPCR

This is really weird, I would not trust such data. How can you amplify splice variants from genomic DNA? That is a complete nonsense. You can only detect splice variants in cDNA.
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