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Standard curve for RT-PCR

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#1 spirallina



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Posted 15 October 2009 - 06:23 PM

Hi all,
I am doing a two-step qPCR analysis of some E. coli samples that have been collected over time from the same reactor. I am using Sybr green for the real-time analysis and have set up a standard curve to quantify the expression of particular genes. In one reaction plate, I only use one set of primers for a particular gene and my standard is composed of that gene (which was PCR amplified from chromosomal DNA and subsequently gel purified). I start with 10ng of DNA for the standard and do a 1:5 dilution. My problem is that my 10ng standard sample begins to amplify right away (and even the 2ng sample). Therefore, when supposedly I have only 0.128pg of DNA, my Ct value is around 15. All of my samples have a Ct value between 23-29 so none of them fall within my standard curve range (which seems to me to be way off). It seems like I have too much starting material for my standard, but I have double checked the concentration and my calculations. I was wondering if anyone else has had this problem or has heard of it and may know what is going on. Thanks!!!

#2 almost a doctor

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Posted 19 October 2009 - 02:10 AM

Hi there, I think you need to further dilute your template. When using purified PCR product as the template for your standard, 0.128pg of template is still a lot of template, as every ng of DNA is going to be of template.
In my case for example, I was quantifying GFP in some samples and I used PCR amplified and purified GFP as template. GFP is ~700bp, which means that in 0.128pg there are 1.65x10^5 copies of DNA, which is a lot of DNA to start with so a Ct of 15 would be perfectly reasonable.

Also, how big is your standard product? I think if using purified PCR product as template, you should talk in copy numbers, as in your actual sample there would be X ng of DNA but it wont all be your gene of interest. If you make your standard curve in copy numbers you can calculate how many copies of your gene of interest there are in your sample.

I find this website really useful to calculate copy numbers without having to do it manually: http://www.uri.edu/r...rces/cndna.html

Hope this helps and makes sense

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