Hello everybody.....
the concentration of my plasmid DNA is 126ng. i want to do real-time PCR taqman probe detection......i want to do serial dilution that should start from 10e8 copy number....so can anybody tell me how to dilute it (how much plasmid DNA and ddwater should be added to make it 10e8). i have attached the calculated values in the attachments. thank you
Hello Mehran Khan,
I assume you have a DNA concentration of 126ng/μl.
The program seems to be telling you that you have 366*10^8 of double stranded DNA copies.
So in theory, diluting it 366-fold (730 μl ddH2O, 2 μl of 126ng/μl plasmid) should enable you to make the 10^8 control concentration. I assume you want to use this as a control for your copy number analyses. In that case, it could be handy to make a serial dilution, by diluting it 2-fold each time.
This should enable you to assign a copy number to the DNA samples you analyze, as well as confirming the correct retrieval of 2-fold differences in your dilution. The Taqman probes should enable you to acquire this specificity, although it might need some optimization. I hope I was able to help you.
Best of luck
i want to make 10 fold serial dilution......using a 10-fold dilution series containing 10e8 to 100 copies/𝜇L. so i am confused how much i have to add ddwater to make it 10e8 copies/ul and then do the 10-fold serial dilution.
thanks for your valuable suggestions
YOur plasmid is currently 2.33 * 10^10 copies/microliter. Dilute it down to 2.33*10^8 by diluting 1:100. I'd recommend serial dilutions. As for volumes, it depends on how many reactions you need to use it for later. Estimate how much you'll need and make twice that volume. Have a colleague or mentor check your math BEFORE you touch a micropipette. Good luck!
Note: most folks use linearized plasmid to create a standard curve. You'll have to set up a restriction digest before you do your dilutions
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