Basic qPCR

[Balasubramani]

What is the minimum concentration of DNA required to conduct qPCR ?

[Mikael Kubista]

If the tube contains a DNA molecule and carrier is used, you will detect it. However, due to sampling ambiguity (finite probability that a test sample taking from positive blood may be negative) the theoretical limit of detection (LOD) by qPCR at 95% confidence is 3 molecules. For real samples LOD is typically lower due to losses in preparation, extraction, RT etc. Using GenEx you can determine LOD for any assay using multiple standard curves.

Good luck

[srujana kapavarapu]

The minimum concentration of DNA that can be used is 10pg which is equal to 3 copies of the gene. you can have a dynamic range from 100ng to 10pg(but the lower limit detection is subjective)

[shangool]

Hi
Do you know how I can convert a PCR protocol to a qPCR protocol?

Thanks
Shangool

[Mikael Kubista]

In theory all you have to do is to run it with a master mix containing syuitable dye such as SYBR. In practice it often does not work well, because traditional PCR assays are usually not optimized sufficiently. You must therefore test the performace of the converted assays. Easiest is to do it with GenEx (www.multid.se) calculating the PCR efficiency and the associated confidence interval.

Good luck

[shangool]

Thanks for your reply. I meant how I can change the annealing, denaturing and extension steps from a PCR protocol to a qPCR protocol?

Thanks

[Mikael Kubista]

PCR cycling conditions do not depend on whether the PCR is traditional or real-time (unless you use Taqman probe). But usually the conditions for the conventional PCR are not optimized enough for qPCR and one may as well start from scratch the real-time PCR assay.

If you are unexperienced in designing qPCR assays, consider joining one of our popular TATAA courses:

https://www.eiseverywhere.com/event_cal ... untid=1690

Good luck!