trouble making a qPCR standard curve

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emmafiori
emmafiori's picture
trouble making a qPCR standard curve

Hi All,
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!
Emma Fiori

Ivan Delgado
Ivan Delgado's picture
Hi Emma,

Hi Emma,

It is commendable that you are trying to get something as complex as qPCR working from scratch without any training. Given that your issue seems to be your DNA standards, here is my recommendation: buy some commercial DNA of known concentration. Knowing for sure that the DNA is of high quality, and of known concentration, would be very helpful in that you can then focus on what is most important: making sure the qPCR assay works as it should, without worrying if the problem is the DNA.

I hope this helps

Curious George
Curious George's picture
 Hi Emma,

 Hi Emma,
Its tough to start qPCR with troubleshooting. My advise is to use spikes when developing new tests/assays. That is an easy way to test performance which will hint you where to start. Various spikes for qPCR optimization are available here: http://www.tataa.com/products-page/quality-control/tataa-universal-dna-spike/.

Good luck