Hi, I have isolated DNA from the 50 mg teeth ground to a powder by using the QIAamp DNA investigator kit (since it contains the protocol devoted to isolation from this type of tissue). I strictly followed the manufacturer’s instructions. DNA was eluted to 25 ul dH2O. The yields and purities, however, were really bad. I was surprised especially by the extremely low 260/230 ratios:
Specimen 39: c = 7.29 ng/ul; A260 = 0.146; A280 = 0.08; 260/280 = 1.82; 260/230 = 0.31
Speciemn 63: c = 20.2 ng/ul; A260 = 0.404; A280 = 0.177; 260/280 = 2.28; 260/230 = 0.83
I will appreciate any suggestions, what could be wrong. Thank you all in advance.
Paja
Low 260/230 ratios may be caused by the chaotrophic salts in the dna binding buffer not being washed away once the dna has bound to the column. Try using less original sample and add one extra wash step to remove the thiocyanate salts before eluting the dna off the column
Paul Rutland
Thank you very much for the quick evaluation and advice. Increasing number of washing steps sounds reasonable. With respect to decreasing the amount of the original sample - manufacturer recommends anything below 100 mg so I decided 50 mg should be enough to get sufficient yield while being safe regarding potential inhibition of the extraction process...
Neverheless, it is easy to test both your advices so I definitely will do so. :-) Thank you.
What are you proposing to use this DNA for? You don't have a lot of it, and this also affects your readings, since it's a ratio: as Paul Rutland says, the chaotropic salts are the problem, but a richer DNA extraction with exactly the same salt contamination would have a much better 260/230 ratio just because the 260 component is higher, not because the 230 component is lower.
The level of salt contamination might be typical for this extraction, with the problem instead being simply DNA yield.
For things like RNA, where you'll typically be reverse transcribing, and for which 'more is better', this ratiometric approach is useful, since it instantly tells you how contaminated your sample is relative to total amount: a sample with a ton of RNA but also a really bad 260/230 ratio must consequently contain a TON of salt, and is therefore bad news even in tiny quantities, while a sample with hardly any RNA and a bad 260/230 ratio has much lower salt contamination, but you'll usually need to use a lot more of it in a cDNA reaction (coz low [RNA]), so it's still bad news.
For DNA, these restrictions don't necessarily apply. For some DNA applications you can get away with, like, "one or two copies of the genome", which DNA at 7ng/ul-1 will be able to supply in abundance (7ng of a diploid 3e9 bp genome is about 1000 copies, I think -back of an envelope calculation).
So, basically: you might be able to get away with using small amounts of your DNA, in which case the salt contamination doesn't matter.
John Hildyard
DNA is aimed for bisulphite conversion and subsequent methylation analysis.
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