I am working on the soil microbiome of heavy metal contaminated soil.
I extracted gDNA from the soil sample using the FastDNA Spin Kit and performed ethanol precipitation to clean up the salt contaminants in my gDNA sample. (so that my 260/230 ratio can be improved).
However, my DNA did not produce an obvious precipitate after incubating with 2 volumes of 100% ethanol overnight. (I followed the protocol from this website: https://bitesizebio.com/2839/dna-precipitation-ethanol-vs-isopropanol/ )
I proceeded with 70% ethanol washing and nanodrop quantification. The concentration was decreased by almost a half and the 260/230 was not increased to 2.0.
May I know what are the possible causes of the reading? What should I do to ensure the purity of DNA? (as both 260/280 and 260/230 ratio of my sample are low)
I would like to know the troubleshooting steps that could be followed other than the suggested guidelines by mp biomedicals.
The nanodrop results are attached for reference.
Thank you.
Hi! Sorry for your troubles - there could be a couple of simple reasons for such results: try using a higher concentration of ethanol (I like to go for 80-90%). Did you remember to cool down the ethanol before adding it?
In my experience that you should incubate at -20C for a minimum of 15 min. I incubate anywhere from 15 min to overnight for smaller PCR amplicons: fragments under 100bp precipitate less well (and with soils samples, I suspect you have a lot of those) than larger fragments with 1/10 vol sodium acetate but the addition of 1ul of 1mg/ml to 20mg/ml glycogen or magnesium chloride to a final concentration of 0.1mM can help - give it a try.
Do not precipitate DNA with ethanol at -80C ( unlike RNA) cause that will encourage excess precipitation of salt and DNA.
You can also try to proceed like that: I never had any problem.
- add 1/10 volume sodium acetate 3M pH5,0 (so 2µL for 20µL DNA)
- then add 2.5 volume 100% ethanol (50µL for 20µL of DNA)
.
- vortex
–20°C for a few hours
- Centrifuge 4°C 15-20 min at 14000rpm
- throw away supernatant
If you have a lot of DNA:
- pellet + 1 ml ethanol 70 %
- Centrifuge max rpm 1 min
- vortex
- eliminate supernatant
- let dry a few mins
- resuspend with water/elution buffer
- vortex and 4°C or few mins at 37°C
If everything fails or you have too many samples to go through, there are good kits for salt clean-up if necessary, like this one:
https://blirt.eu/product/extractme-dna-clean-up-kit/
good luck!
the ideal would be to carry out the dna extraction with protocol using magnetic beads, for example the thermo kit, then you would get a good material.
Everything can vary according to the extraction kit, the pH that it leaves the solution, the chemical compounds present in the medium and a number of other factors ...
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