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Thread: real time PCR melting curve

  1. #1 real time PCR melting curve 
    Forum Freshman
    Join Date
    Nov 2008
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    Hi.
    I tested a primer on real-time using both - genomic DNA and cDNA as templates.
    Everything works but the melting peak temperature for fragments amplified from the genomic DNA is a little bit lower than for the fragments amplified from cDNA. The difference is 0,3 C

    How do you think what could be the reason?


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  3. #2  
    Forum Masters Degree samcdkey's Avatar
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    Jun 2006
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    As you add more and more template, your Ct decreases as it overcomes the background and reaches the threshold faster. It will also decrease efficiency. Have you checked efficiency between the two samples? Perhaps there is too much template in the case of cDNA?


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  4. #3  
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    In fact the amount of cDNA sample was very low (10 - 20 ng per sample). Does the real-time PCR reaction efficiency really have an influence on the melting curve? I mean if the product is the same, the melting curve peak temperature should be the same too.
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