Qubit library quantitation variations

[Smriti]

Hi All,

I am facing lot of variations in my library quantitation using Qubit dsDNA BR assay kit. Lambda DNA stock concentration which is provided by Sigma has a documented concentration of 400ng/ul and Qubit shows above 550ng/ul. What might be the root cause? Is it pipetting error or people tend to get variations from sample to sample?

Please advise!

[Gina_P]

Hi, it could be pipetting errors, but more likely you simply have a bit of batch variation from Sigma so that it's not exactly 400 ng/uL. However, I don't have any experience with these stocks so anyone else can comment if they know.

[lterhune]

Hi,

In our experience the Qubit BR assay is less accurate than the HS assay, so try doing a dilution of your DNA and using the HS kit if you have it. Our BR assay usually read higher than the HS, even if the stock DNA concentration fell within the range of both.

One QC thing we've started is doing a spec of your Qubit standards, especially standard 2. We had one kit with problems and found it was almost 20% above the concentration written on the tube.

Also mix your samples extremely well, this can make a huge difference especially if you're only measuring 1ul.


-Liz

[mmmm]

Qubit (HS, BR)

when I used Qubit HS (high senstivity kit) for measuring gDNA conc., I got entirely different concentation compared to BR (broad range) kit. do you know, what is the difference between Qubit HS kit and Qubit BR kit for DNA conc.?

[chevrm]

When was the last time you ran the standards? If you're not running them every time then you're pretty susceptible to pipetting errors in your Q-reagent/buffer sln.

[mmmm]

I run the standard everytime and Qubit reads the right conc. for the standard but I got different readings for the same sample (20ng/Ml when HS kit was used) and (80ng/Ml when BR kit was used), have tried on other samples too and get different readings- it is not pipeeting errors so what is the explanation?

Also, would DNA concentration decrease that much following freezing and thawing?