I am using a Qubit to get the concentration of DNA stock (extracted from tissue). It gave me different concentrations for the same sample. 50400 ng/ml a couple days ago vs. 15200 ng/ml today. The qubit was recalibrated each time and I’m not sure if that had something to do with it. Im using these concentrations for absolute Quantification PCR and I’m worried this will effect my unknown sample concentrations
This cannot be the systematic error (small variations) and definitely is the user handling or pipetting error.
Make sure your sample is well mixed so that you get a representative sample in your qubit reaction. As I doubt pipetting error would cause such a drastic reading change.
And to double check this second reading wasn't the same Qubit reaction? Once the dye and buffer are combined, the recction is only stable for 2hrs
Qubit is most sensitive so far I know for the quantification of dsDNA or ssRNA.The chance of degrading your cDNA or DNA is very rare if you stored in recommended temperature. Please check the following before the work:
1. Quality of your reagents and place where you stored (avoid from light and heat, store at -20 deg C)
2. Pipetting error may cause variation in result value.
Romen Naorem Qubit kits (DNA BR, HS, RNA BR HS, Protein) are all designed to be stored at room temp. and the standards in the fridge (4C) for six months, storing at -20C may actually be damaging the kit through repeated freeze-thaw. The only kit that is different is RNA IQ which is -20C and -80C for components and standards, respectively. Also, I meant to put 3 hours for stability once combined
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