Hello.
I am currently extracting genomic DNA from fungal cultures (filamentous fungi) using a BioVision Genomic DNA Isolation kit.
However, when the fungal colonies are pigmented, espectially with dark pigments (blackish or greenish), these pigments are always co-extracted with the DNA, which ends up giving weak A260/280 and A260/230 ratios; thus decreasing, significantely, the purity of the DNA extracts which are intended afterwards for PCR.
Can anyone help me with a way to remove these pigments before final elution of the DNA?
Thank you.
We use the protocol in this paper and we made RT-qPCR on filamentus fungus without problem.
Do you try to culture it on liquid culture?
Do you perform the Phenol/Chloroform and Ethanol precipitation steps that are stated as optional in the protocol? These steps should get rid of you pigmented materials, assuming it is heavy cell wall material.
@ Norman van Rhijn.
No Sir I do not perform these steps.
Thank you for your answer.
Dear colleague,
Sometimes you do not need to extract all pigments (for example, melanins) in order to obtain optimal PCR results. PCR reactions tend to be easily affected by pigments, but they are not so easily affected by dilutions of DNA. I used to perform PCR from dematiaceous fungi, and one easy way to avoid inhibition by pigments was to perform the test with series of dilutions of DNA (in water). You might try, for example undiluted DNA, and diluted 1/20, 1/50, 1/100, and 1/1000. For multicopy regions, such as ITS of LSU, even highly diluted DNA should work well, but pigments should not interfere anymore.
In addition, take into account that pigmented fungi often produce much smaller amounts of pigment when grown on media enriched with simple sugars, such as on Sabouraud-dextrose agar, compared to other media such as cornmeal agar or oatmeal agar.
Success!
@ Laith Al-Ani
Thank you very much for your answer. As mentioned above, I am using a commercial kit for the extraction, BioVision Genomic DNA Isolation kit. But it did not remove the pigments.
You can dilute the DNA, I think this will be dissolved your problem .....
Yes, this could be another way to do it, if everything else fails.
Thank you very much Donya Nikaein
You might use a purification product by magnetic beads to purify your extraction. https://www.thermofisher.com/br/en/home/brands/product-brand/dynal.html?s_kwcid=AL!3652!3!352542913364!b!!g!!%2Bmagnetic%20%2Bbead%20%2Bextraction&ef_id=CjwKCAiApOvwBRBUEiwAcZGdGKqstope4lzYSDcNenvZAaGNRg85Ze7We9v2H962zwsuH_C4W9UTWRoCSeUQAvD_BwE:G:s&s_kwcid=AL!3652!3!352542913364!b!!g!!%2Bmagnetic%20%2Bbead%20%2Bextraction&gclid=CjwKCAiApOvwBRBUEiwAcZGdGKqstope4lzYSDcNenvZAaGNRg85Ze7We9v2H962zwsuH_C4W9UTWRoCSeUQAvD_BwE
As you have already been told, diluting the DNA may help, however I have had the same problem and changing the extraction method was a big improvement (CTAB extraction from 24h liquid cultures). In addition, precipitation with NaAc 3M pH=5.4 and EtOH as final steps of the extraction also contributes to obtaining "clean" DNA.
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