ds HS Qubit assay showing variability

[Smriti]

Hi folks,

I am getting lot of variation in my genomic DNA concentrations when I measure it using HS assay. I always warm the DNA for 10 minutes at around 40C to improve its homogenity. Then I diluted the stock to around 4ng/ul since the Std 2 of HS assay is at 10ng/ul, so to be in the range of kit I choose this concentration.
I was getting the concentration in the range of 8ng/ul instead of 4ng/ul. The values on my standard are: Std 1: 129.14 and Std 2: 28377.09.

Any suggestions on getting less variability in the data? Is this value on Std 2 signifying some problem in Std itself?

Thanks!

[darwin142]

Hi Smriti,
It has been a while since you posted so I expect that you probably have the answers but in case you don't:-
When preparing the standards for the calibration you add 10ul of the 10ng/ul which means the range is a lot higher. In fact it will measure up to 125ng/ul if using 1ul of sample in the 199ul of buffer.
When I use the qubit I prepare the standards as per protocol and I also add 1ul of the second standard to 199ul of the buffer to check it reads 10ng/ul.

The kit is HS but at very low concentrations you would probably need to add 20ul of sample to reduce variablity.