I am very new to qPCR, and I have no one teaching me so I've had to learn everything myself from every resource I can find, so please forgive my ignorance. Nearly all my molecular experience is self-taught or following pre-made protocols, and I've now found myself running a micro lab with no idea how to design experiments. So I need lots of help!
I am trying to make a standard curve for qPCR of enterococci. I've extracted the gDNA from the reccomended strain, quantified it using a spec, and did all the calculations for serial dilutions. But when I started checking my freshly made standards on the spec, I was getting very bad numbers (10^8 when it should have been 10^1). I managed to get the higher concentrations (E7, E6, E5) confirmed on the spec as close enough (how close is close enough??), but after multiple faileld attempts, I gave up on the lower concentrations.
Any suggestions for what I'm doing wrong, or how to get more accurate serial dilutions?
Any info is greatly appreciated!
Thanks so much!