Making a qPCR standard amplicon from cloning, why? - (Sep/06/2012 )
Just a quick one that I am hoping someone can answer:
I am interested in quantifying the number of bacteria in a population using qPCR. I have primers to a gene that is present once per genome (DNA gyrB) and amplifies up a 150 bp fragment. I will make a standard curve using purified and quantified PCR amplicons for this assay.
My question is that I have read papers in which people go to, what I consider to be great lengths to obtain template for their calibration curve by cloning the fragment into a plasmid and then extracting and purifying it this way. Why do that when you could just PCR up your product, verify via a gel and purify and use it that way? It's making me question just using PCR product for this.
If you want to do a standard curve for qPCR you need to know the total number of template molecules that you have from the beginning, and amplifying them in the same way and same efficiency that in your test sample, that is why it is better to use serial dilutions of a known number of plasmids (real number of molecules) or PCR fragments containing your sequence of interest, and use them to amplify the same amplicon (same sequence) that you are testing.
I hope this will help you.
Thank you Akdor. I have performed this assay and it has worked fine, I am just confused why some people go to the trouble of cloning. I know the molecular weight of my amplicon, and I can quantify how much purified product I have from a regular PCR so I can calculate the number of copies in my PCR-generated standard - i just don't see why you'd bother cloning it and using a plasmid which you'd then quantify and do the same calculation on anyway. Do you mean that it would achieve a more similar efficiency being plasmid associated than just a short amplicon? I can see that. Hmmmmmm!
For an absolute quantitation you do just once or so and you don't want to bother with cloning, using purified amplicons as standards is a way. You should run them on gel, gel extract, clean up and quantify and then calculate the copy number. It's better to mix it with some dummy nucleic acid, but since you work with PCR product anyway, maybe it doesn't matter. You need to use the standards fresh, they degrade while in storage.
If you intend to use it regulary, invest into TOPO cloning. Ligate your product to selfligating plasmid either TA version or Blunt with killer gene (Invitrogen sells those for example) in 5 minutes and very high efficiency. You can buy kit together with cells. Then you purify plasmid, quantify, calculate copy number and dilute with E.coli 18S 23S RNA from Roche as a dummy NA and you can have your standard almost forever.
The second biggest problem apart from PCR products degrading from ends is that low complexity templates amplify differently than real template. That's why the dummy RNA is used.
Thank you both very much. That all makes sense.