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About serial dilution of cDNA were amplified by real-time PCR


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#1 baopingtcm1984

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Posted 17 February 2011 - 09:43 AM

HI,ALL

i am gona do real-time PCR ,but main question is to identify the efficiency of amplification about both target gene and internal gene.

so,i want to use cDNA dilution to get amplification efficiency. Now, how to do serial dilution of cDNA? THANKS.

#2 ivanbio

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Posted 17 February 2011 - 10:05 AM

Preparing a serial dilution of your cDNA is straight forward. Aliquot some of your cDNA in a tube and call this "undiluted". This is the first point in your dilution series. Then take 10 ul of this cDNA and transfer it to a second tube. Add 90 ul of water to this second tube and mix. This is your 1:10 dilution. Now take 10 ul of the cDNA dilution in this second tube and transfer it to a third tube. Add 90 ul of water to this third tube and mix. This is your 1:100 dilution. Continue doing this until you have at least 5 dilutions. This is your serial dilution. If your assay is very sensitive, and have plenty of cDNA in your undiluted cDNA, you should be able to detect a signal in the 1:100,000 dilution. You can of course go even further and dilute your cDNA even further. There is no limit other than how sensitive your qPCR assay is.

Good luck

Ivan
Carlsbad, CA




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