How to prepare serial dilutions of RNA or cDNA - (May/16/2013 )
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?
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.
Some people said that pipetting RNA or DNA can cause damage, so I avoid it. From your suggestion, it seems that pipetting is ok.
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.