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Making a standard curve from bacterial suspensions

qPCR DNA extaction

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#1 firefly2280

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Posted 19 January 2013 - 10:37 AM

Hi

Sorry if this is an obvious question but Ive just been confusing myself!

I want to make a standard curve for S. aureus to run on my qPCR. I have bacterial suspensions - say 10^8 / ml.

If I just do a bead-beat and lyse the cells, proteinase K treat then etc, then I have a suspension that I can say is 10^8.

But then if I add 10ul to a qPCR, im only actually adding 10^6 gene copies, right? So if I want to do it this way, I need to start at a much higher dilution?

Is this right or am I totally off the mark?

Then, if I get 10ul of sample come up at 10^6, does that mean I have 10^6 in my 10ul of sample!?

thanks so much

#2 pito

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Posted 19 January 2013 - 11:13 AM

I dont really understand your question/problem.

If you have a 10^8cells/ml suspension and you use 10µl of that, then you have 10^6 cells in total (in your 10µl).
If you add this to your PCR then you would indeed have 10^6 gene copies (accepting the fact that you only have 1 copy of that gene per genome/bacterium, which I dont know).
Also: you have 10^6 bacteria in your 10µl, but you will not be able to recuperate 100% of the DNA anyway... so you will not have 10^6 gene copies anyway...


So what is your problem? You want 10^8 copies of your gene?
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.





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