We have done some RNA seq libraries and quantified the final product in nanodrop. As always, we blanked with the used solvent (0.1 TE buffer).
Most samples are really good, around 6-20ng total DNA
But two samples showed negative 230 and 280nm curves. We are unsure about what this could mean as it seems to make no sense. We even repeated the measure of one of those samples and it gave similar results (negative curves, but positive 260nm)
These are the images
Example of good sample
Negative 230 and 280nm samples
It means your absorbance at those wave lengths are lower. Does your library contain EDTA? The EDTA absorbs in that region, especially at 230, so blanking with TE sets the baseline high. Any sample after that lacking EDTA will probably be negative. Try blanking with the same buffer as your library. Just a guess.
We used the same blank as the last step in the kit (TE), which contains EDTA. That's why we don't understand what happened. These two samples have been eluted in TE as well (100% certain).
UV absorption is not the best method for quantifying low concentration DNA. Better use fluorescence based method. EDTA interact with multicharged ions used in the binding substance, resulting in residual absorption in the UV range. If you use UV absorbance method, your blank buffer should be submitted to the same last mock steps.
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