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How to prepare serial dilutions of RNA or cDNA


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#1 xxcici

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Posted 16 May 2013 - 12:51 PM

For making standard curve using real-time PCR, how to prepare 10-fold serial dilutions of RNA and cDNA? I tapped tube 200 times to make sure that the sample has been mixed very well. Then my standard curve is not with a good slope. For each dilution, the amplification curve is very beautiful. Then, I guess the problem is from dilutions. Are there some other points that can affect amplification?

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  • Amplification Plot.jpg

Edited by xxcici, 16 May 2013 - 12:51 PM.


#2 phage434

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Posted 16 May 2013 - 02:41 PM

Genomic DNA is quite viscous and non-uniform in concentration within a sample, no matter how well you mix it. I'd suggest that with those samples you reduce the viscosity by mechanical shearing or cutting with rare cutters. Often forcefully expressing the sample through a small diameter needle several times will shear the DNA effectively.

#3 xxcici

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Posted 16 May 2013 - 05:18 PM

Some people said that pipetting RNA or DNA can cause damage, so I avoid it. From your suggestion, it seems that pipetting is ok.

#4 phage434

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Posted 16 May 2013 - 06:34 PM

Well, that is correct, it will damage the DNA. But in this application (and in almost all), you don't need very long strands -- they need only be long enough to contain both ends of your amplified region. Very long strands cannot be diluted accurately.




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