I've completed a genomic DNA extraction using the ThermoFisher Yeast DNA extraction kit on some Basidiobolus ranarum fungal cells. Of note, before performing the ethanol wash for this protocol the pellet for both samples already appeared white. Upon completion I observed a white precipitate that would not resuspend, even after 24 hours and heating at 65ºC for 30 minutes.
I carried out a nanodrop on two samples which gave the following results (tubes were very briefly spun down to collect precipitate at the bottom):
1. Concentration: 130ng/µl, 260/280: 1.78, 260/230: 0.82
2. Concentration: 97.4ng/µl, 260/280: 1.69, 260/230: 0.69
Given the low 260/230 ratios I suspected that there was some contamination of some sort, which could be giving false readings for the concentration.
I carried out a broad range DNA Qubit assay and got the following results:
1. Concentration: 1.20ng/µl
2. Concentration: Too low
Ultimately my question is: Is it possible for Nanodrop readings to be this wildly different if there's signs of contamination?
In the past I've experienced issues with quantifying genomic DNA on the Qubit. Is it possible the Qubit is reporting inaccurate results or is it more likely it's just a big contamination problem?
for some "dirtier" samples there could be quite a significant disconnect between Nanodrop and Qubit (the last one being more "precise"). but I've never seen such a dramatic difference
Hi Gina,
Nanodrop is not accurate in estimating nucleotide concentration unless the sample is very pure. And it is not surprising that it can overestimate the concentration by 100x, especially in your case the absorbance readings (which Nanodrop is pretty good at) indicate there are contaminates in your samples.
I would recommend running your sample out on a gel, side by side with a ladder for which you know the band masses, to estimate your DNA concentration. This way you can have a pretty good idea of the quality of your sample too (e.g. whether there is any degradation).
Qubit is the choice if you want more precise quantification. If you expect your DNA concentration to be low, use the "High Sensitivity" option of the Qubit.
Hope it helps. Good luck!
Best,
Sheng
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