Alternative phenol-free method for DNA extraction - (Jan/30/2014 )
I am conducting DNA extractions from butterfly tissue. I have tried using a salting out method and an adapted Lund protocol for extraction,
but my curves in Nanodrop still aren't right (indicating some kind of contamination, like salt or EDTA, and in some cases protein).
I am looking for a suitable protocol that does not involve phenol extraction, as this would be more expensive to us (we would have to setup
special safety equipment and buy expensive chemicals).
The reagents we have, and would ideally use are: EDTA, SDS, Tris, NaCl, NaAc, and ethanol. We are looking to add another step to eliminate
the proteins from our samples.
Any help would be greatly appreciated (even more so if your opinion is supported by a published paper).
Alternatives would be kits (expensive, except you recycle the columns and prepare your own buffers when empty), glass milk based, or chelex (just to mention some).
For insects I also used a lot a salting out protocol, but usually adding Proteinase K for the lysis step. Also additional washing steps help to improve purity of the DNA (though you'll lose more DNA then).
Question is of course if you need very pure DNA for later applications, for standard PCRs usually salting out is enough.
edit: I used a protocol in this paper quite often:
Sunnucks, P. & Hales, D. F. (1996). Numerous transposed sequences of mitochondrial cytochrome oxidases I–II in aphids of the genus Sitobion (Hemiptera: Aphididae). Molecular Biology and Evolution 13, 510–524.
In most phenol:chloroform protocols you can avoid the phenol. Phenol mainly increases the protein removal efficiency, so you may need to repeat the chloroform extraction at least one more time after the supernatant looks transparent.
I've used this protocol, slightly modified, to extract high molecular weight DNA for genome sequencing from fungi and I got good results right the first time I did it. And it was the first time doing a chloroform extraction.
It is a CTAB-based protocol