Dear all,
I am facing Subit issues, for mosquito genomic DNA measurements (for gDNA library preparation for NGS).
The same sample was measured by 3 different people (in december 2015, august 2016, and february 2017). We all get different concentrations, and from what I can see, the DNA concentration is always increasing (3.5 - 12 - 45 ng/µL for one sample, for example).
Do you have any idea of what the problem can be? Is it due to dye stability? We keep them at RT, in a black plastic box. For 2015, it was another kit, but I had the same for 2016 and 2017. Also, we don't have special air-conditioning, we might have had some temperature fluctuation (at least between summer and winter, like 30 - 20°C in the lab).
Thank you in advance for your interesting comments and suggestions!
Impossible to have a 10-fold change, these are 200-µL samples and I still have 200 µL in my tubes...
If the evaporation is not the case.
Which type of kit did you use? A broad range or a high sensitivity? Did you perform a calibration before an each set of measurements? Did you use every time the same sample volume for the measurement? How many microliters?
@Artur Burzynski
In the case of DNA degradation, I would expect a decrease of the concentration on Qubit.
Samples were kept at - 20 of course. The device is the qubit 2.0 and it is a dsDNA HS kit. I do calibration for every new measurement. I measured with 1 or 2 microliters. I even have differences with a same sample measured with 1 or with 2 microliters (I did two measurements with the the same DNA, and indeed entered the right volume in the machine software..
At least two microliters are recommended for the measurement on Qubit. It is better to use the same volume everytime to be able to compare concentration.
Kit can be stored in RT but calibration standards should be placed in fridge.
DNA samples should be tempered on room temperature and properly vortexed before measuring.
Is your Qubit placed in pre-PCR room, only DNA samples were measured?
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