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Old 03-04-2015, 12:11 PM   #12
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Location: Chicago, IL

Join Date: Dec 2014
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Originally Posted by Terminator View Post

Did you re-run the undiluted DNA side by side with the diluted DNA as a control?

What do the raw fluorescence readings for controls look like between the two runs?

Assuming you have a homogeneous mixture of DNA with no clumping, try adding additional Qubit standards. I have seen a fair deal of variation in the past so now I run 10 controls (2 x each of 0, 25, 50, 75, and 100 ng) and plot it myself to calculate my DNA concentrations.
I did run undiluted controls a couple of times but should start doing it every time. If I run undiluted controls over and over (without changing anything) there variation of maybe around 5–20% or so (which I have no problem with, at least it's qualitatively similar). The larger error comes in when we change the dilution and especially when we concentrate the DNA to a lower volume but then it is more viscous—so I imagine as suggested above that this is a serious source of error.

That is a great idea to do the extra standards across the range and then plot- thanks!
Myrmex is offline   Reply With Quote