I have checked a sample of salmon DNA (provided as 'solid' dsDNA and brought into TE-buffer up to a certain concentration) with the Nanodrop. According to the measurment (performed several times), the sample should contain 50 ng/uL of DNA. However, when quantifying the sample with the AccuClear kit, I go only 20 ng/uL. The sample is stored at -20 C and has never been exposed to high temperatures (only RT). Due to freeze and thaw cycles and maybe some UV-light, I would expect a concentration a little lower than 50 ng/uL. But 20 ng/uL I cannot explain. Now I would like to check if my sample indeed contains a lot of ssDNA that is messing with my Nanodrop measurement.
It is not uncommon to see concentration differences between different analyses. The nano drop isn't really the best method for quantification. Additionally, single stranded material and mono nucleotides are extremely absorbent, so you'd expect your concentration to go up over time if there was degradation not down. It is possible that your sample has a small amount of nucleotides present and is causing the discrepancy between the two methods.
Dear Matthew,
I know that nucleotides and ssDNA also contributes to the absorption value at 260 nm and that the Nanodrop is therefore not the best method to quantify my dsDNA. However, it is just 'pure' DNA and not from an e.g. PCR mix, where you would expect the prescence of nucleotides or smalle fragments of DNA (primers). The DNA might be somewhat degraded, but then I would not expect a difference of 50 ng/uL versus 20 ng/uL.
Brigitte
Dear Brigitte
I think we just do gel migration to verify because the ssDNA and dsDNA are not the same size.
Best
Innocent
That is a possiblity, but I don't know the exact lenght of the DNA (should be around 2000 bp according to other literature, but it is not determined by the manufacturer) and I am also not sure if the DNA is all of the same lenght (especially when indeed degradation took place). However, I still want to give it a try.
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