First time Real TIme - (Jul/16/2008 )
Hi all, I am dealing for the first time with Real Time PCR....My background is environmental Engineering, so my knowledge of Molecular Biology is very limited. I have some environmental samples extracted DNA and I have to run Real Time with them. What are the steps that I have to follow? I first of all need some advices regarding how to build my calibration curve. Can I just extract DNA from an Ecoli culture and do serial dilutions with that ? Do I have to know the DNA concentration of the DNA that I have extracted?
I have a iQBiorad, and I don t also understand what is the real concept behind the copy number. How can I know the initial copy number in my sample?
If I don t have the chance to measure DNA concentration, what can I do? How can I approach the calibration curve?
Thanks a lot.
first of all you should know which gene or organism sequence you want to quantify. Then design primers (and probe for TaqMan assay) with an appropriate software.
Then you have to decide which quantification method you want to use:
-absolute quantification (then you need your standard curve)
-relative quantification (delta delta ct method or other)
Then you have to set up your standard or calibrator.
Standard - easiest way: you take an external std. which can be a purified plasmid with your target sequence (gene), as you can determine the conc. of your plasmid and know the size, you can calculate the molar conc which gives you the copy number, then make a 1:10 dilution series e.g. down to 1 or 10 theoretical (!) copies.
The delta Ct between your 1:10 dilutions should be 3.3 (range 3.1 to 3.5) which is a good efficiency. If you don't get a good efficiency, try to optimize your assay.
If you have established your std. curve you can try to measure your samples. Before it doesn't make sense.
However, don not expect too much for the first few experiments. It needs some time to learn how to do good real-time PCR (qPCR) experiments. So maybe you start with running only an assay and samples you get from a experienced collegue. Then you know if does not work that it is you, not the assay or your sample.
As you want to perform experiments with E.coli DNA I warmly recommend you to use a master mix from the 2 top companies: Roche Applied Science or ABI. Let me explain you the background: all master of course contain a polymerase. These recombinant proteins are expressed in E.coli strains and with the enzyme there are small amounts of dna co-purified. Only these 2 companies have a well established method to clean up their enzymes that the dna is removed effectively. This is usually not a problem when working with human or animal targets but becomes important when working with E.coli samples (I think you understand).
As ABI only has master mixes for their instruments, only Roche offers masters which fit your iQ5 instrument. So use e.g. the FastStart Universal SybrGreen Master (Rox) for Sybr assays or FastStart Universal Probe Master (Rox) for TaqMan probe assays. They both work very well on the iQ5 which I know from my collegue who is really into real-time PCR. If you decide to work on qPCR more intensively I can recommend you the Universal Probe Library - but this is really for more experienced users.
However I can only remind you that qPCR is not a simpe thing. So best is if you look for someone experienced in this field to give you some support!!!!!!!
Maybe you start reading with the pcr manual from roche. They have a nice chapter about real-time pcr. Here is the link:
If you have further questions let me know….
Thanks a lot for your reply.
I want as first step built my calibration curve. So I have DNA extracted from Ecoli (pure culture) and I bought SYBR green from Biorad. I also have two universal primers : the 357F and the 518R.
Tell me if it is the right way to proceed:
- determine by spectrophotometer the concentration of DNA from the extracted Ecoli sample.
- then I know the genomic size of the ecoli : around 4600 kbps
- and I can calculate the mass of the genomic size and from my calculation is around 5*10^-3 pg
- I also want to have a series of samples from 300000 to 30 copy numbers ( so I basically multiplied the expected copy number for the mass of the genome size and I obtain the mass of DNA needed to obtain that copy number) . based on this last number I dilute my samples consequently.
- from that I can define my standard curve copy number-ct
then if I also run a unknow sample, I just need to read the ct value related to this unknow and read the concentration back.
This is what I understand. And also if what I am saying is right, why people complicate their life looking for plasmids instead of just extract DNA from a pure culture and measuring from that the concentration?
Thanks a lot.
I will also read the document that you suggest. Thanks again.