How to generate qPCR standard curve in excel and calculate PCR efficiency

A standard curve can be generated using a 10-fold dilution of a template amplified on a real-time system (Example: ABI 7500). Each dilution can assayed in triplicate(its always better to do in duplicates or in triplicates).

[Othmanbaradwan]

simply explained without speaking, well done!

[graemeblunn]

Thank you very much, efficiency calculation really helped me out! Your method is different from my profs... I tried using his and was getting efficiency of over 3000%, using your method, the numbers were much more realistic, 93%

[kenv]

How do I know the copy number of the cDNA? I just did a serial dilutions. How to fit the dilution levels to the plot?

[orlandoducati]

I have a question, if the mean of my ct value is 27.6. When I plug this number into my standard curve equation to generate DNA concentration, the concentration is like 660, which doesn't line up with my standard curve plot.

[vikasfandade]

how is your Ct value so low when you have highest dilution? (low Ct values means high initial concentration of DNA, and at higher dilution how can you have higher initial concentration?)

[mercedesjimenez]

Hello, I am trying to develop your method, but I can´t because I have a low slope and I don´t know how to solve it. Thank you!

[fara.r.]

Copies/mL? Not copies/uL (microlitre)?

[Ahoooooody]

i did the standard curve as u did following every step and I got the pcr efficiency 110% is it realistic

[omar]

Not sure I can quite follow what you want to say? And how could your Ct Value is so high, while your dilution/Copy number is very low?

- Low dilution >>> low Ct value>>> high copy number.
- High dilution >>> High Ct Value >>> less copy number.

Could you please clarify it a bit further?

Thanking you !