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I prepared genomic DNA from pig stools but the concentration was very low — about 3~4 ng/µl, when I measured it using nanodrop. The A260/A280 and A260/A230 values also did not come out very well (0.4~0.5 and 1.0, respectively). I also tried filtering the DNA two times but it didn't improve the quality. Does anyone have experience with DNA extraction from stool samples and suggest me how I can improve the quality and yield?

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Expected photometric ratios for pure DNA are around 1.8 (A260/A280) and 2.0-2.2 (A260/A230). For reference, if you are extracting pure RNA, its ratio is ideally 2.0 (A260/A280).

Often, lower ratios usually indicate low purity due to contaminants or simply a very low amount of DNA.

EDTA, carbs and phenol among other chemicals all have absorbance at 230nm. If you used TRIzol reagent to extract DNA, you may also get some absorbance from this reagent at 270nm if you incorrectly pipetted some of the phenol phase (pink stuff) or the interface (white cloudy stuff between the aqueous phase and the pink phase). If you measure a low ratio, like you did, it probably means that in your final DNA extract, you seem to have (a) things that are not DNA in your solution, and/or (b) a very low concentration of DNA.

If you suspect you have enough DNA, you can purify to try to remove non-nucleic acids. However, with ratios that low, I suspect you should extract DNA from scratch; it does not appear that you have enough to rescue simply by purifying again.

Which method did you use to extract your DNA? What do you mean by filtering?

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  • $\begingroup$ Oh I didn't use trizole reagent. I used a commercial kit. filtering means DNA binding and eluting at column tube during experiment. $\endgroup$ – Koreanraichu Jan 10 at 0:51
  • $\begingroup$ You're using spin columns, I'm sure. They contain a silica resin that selectively binds DNA (RNA is also possible), by varying salt conditions. You pass the solution with your DNA by centrifuging, the DNA gets trapped on the resin, and then elute the bound DNA with a second pass of an elution buffer. It's quite a high-quality way of purifying DNA from a solution, in my hands it works better than TRIzol extractions. I have no idea how you obtain an aqueous solution workable with columns from pig stool. In principle, it should work well if the input is good enough. Sorry that's all I got! $\endgroup$ – S Pr Jan 10 at 16:19

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