I have performed many chromatin extractions, but at the end of every experiment I have a large peak at 220-240nm when measuring DNA concentration with a nanodrop. I have had some very limited success by including diethyldithiocarbamic acid (DIECA) and pvp-40 in all of my solutions, but the peak remains. I assume that it is DNA oxidation and not polysacharides because multiple ethanol washes through a cellulose column does not eliminate this peak from the spectra. Anyone have any suggestions
This is more complex than simply adding some reagent. Under some conditions adding and anti-oxidant (reducing reagent) can actually accelerate oxidation. It has to do with the whole buffer system. Simple example in reference (link).
http://www.ncbi.nlm.nih.gov/pubmed/9443069
please send your questions to my student:
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Have a nice day! Fangpu
Hi Rick,
It doesn't sound like tannin to me - when these interfere, they bind proteins and nucleic acids as soon as the cells are lysed. This prevents their purification by precipitation or column-based methods. However, with our tannin-rich (or other polyphenol-rich) samples we use DNA extraction methods that have a decent amount of PVP and CTAB in it, and also a reducing agent such as DTT or 2-mercaptoethanol. (For RNA, we typically use a kit, but supplement the lysis buffer (450uL) with 100 uL of a solution containing 20% PVP and 20% CTAB (it is hard to dissolve, and needs to be kept warm!)).
Have you run any gDNA out on a gel to check it? With spec quantifications, a number of things can interfere - guanidine salts, alcohol, phenol etc. This link might be useful:
http://seqanswers.com/forums/showthread.php?t=21280. Perhaps the peak is from the DIECA (see link... DTT also has an absorption peak, pehaps DIECA does also)? I would check your DNA on a gel to ensure it looks to be high molecular weight. Then try digesting a sample - that is the best way to ensure it is suitable for downstream applications. For quantification you should use a fluorometer (e.g. Qubit), as we have found UV-spec unreliable for gDNA quantification, in part due to some of the extraction components (CTAB,+ others). The 260/280 260/230 ratios are still useful to check if there are other contaminants in the sample.
Happy to send you the protocol I've used, if that is any help - but I would check your samples first, before discarding them.
Good luck!
Nick
There could be some organic compound contamination in your sample. Just carefully wash with buffer solution containing antioxidant and nuclease inhibitor.
best wishes
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