I am assisting with a student research project in which they are trying to identify antibiotic producing bacteria in soil. They are growing isolated colonies of the bacteria on TSA plates and then trying to extract DNA for use in PCR of the 16S region for eventual sequencing. The bacteria are both Gram positive and Gram negative mostly bacillus shaped.
They have tried several different methods for DNA extraction: Chelex, Chelex with ethanol precipitation, Chelex with Proteinase K, and freeze/thaw with Proteinase K. Regardless of the DNA extraction method, the 260/280 ratio (song a NanoDrop Lite) of the soil bacteria is around 1.2-1.3; the best has been 1.43 using the freeze/thaw/Proteinase K method. The lab strain of E.coli that we use as a positive control consistently yields a ratio of 1.7. I am not sure why there is such a difference between the soil bacteria and the lab strain E. coli when they are extracted at the same time using the same method.
Any suggestions that I can give to the instructor in charge would be greatly appreciated.
Dear Kendra Kimberley,
There are some crucial steps in the procedure of DNA isolation from the bacteria. which decide the quantity as well as quality of the isolated DNA.
Kindly go for proper lysis procedure by increasing the incubation time. Please go for repeated PCI treatment during the extraction procedure.You can also adopt warm elution process for the elution of the DNA.
Soils are notoriously difficult to work with because there are a lot of polysaccharides and humics that can co-extract with the DNA. My impression has always been (and it might not be correct) that Chelex-based extraction is really just for pure cultures, where you have no significant environmental contamination.
I don't know what chemicals you have access to, but you could try using an approach similar to the one I've attached, using phenol/chloroform is often a great cleaning step. We've also found sometimes it's beneficial to use polyvinylpyrrolidone (PVP) or PVPP in your extraction buffer to remove humic acids.
http://aem.asm.org/content/66/12/5488
Thank you both for your responses. I will pass on the information. To clarify, the bacteria we are trying to extract DNA from are growing as pure cultures isolated from the soil. This is what is most perplexing since we can isolate good quality DNA from our lab strain E. coli but not from our pure cultures of soil bacteria.
Sorry, I didn't read the question correctly. The phenol/chloroform step would still be a good idea, though.
Dear Kendra Kimberley,
I think that the low 260/280 ratio of soil bacteria might be influenced by the Gram positive spore forming bacteria present in your cultures (Firmicutes such as Bacillus and Clostridium species, etc.). Spore-forming bacteria are more dificult to extract and to purify. So, it is not surprizing that the freeze/thaw/Proteinase K method lead the best results. It may help to: add a Lysozyme treatment for the lysis step, increase the incubation time for proteinase K; add another phenol/chloroform step for cleaning; add another 2 cleaning steps only with cloroform; 2 steps with ethanol and increase the drying time of your matrix before elution.
Since the abnormal 260/280 ratios indicate that the sample is either contaminated by protein or a reagent such as phenol, repeated cleaning steps might really work. Although purity ratios and spectral profiles are important indicators of sample quality, the best indicator of DNA quality is functionality in the downstream application of interest such as PCR. So, abnormal 260/280 ratios might still lead to good PCR amplificatios.
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