I have tried some protocols in order to extracting DNA from Filamentus fungi, such as proteinase K, RNxplus, TRizol, 95 Degrees of c. heat and ....
but still its a failure. how should I extract DNA?
thanks in advance.
It seems that I could not extract DNA because of cellular wall... is it possible to break down this wall by heat and then follow the process with Proteinase K protocol?
what could work for you (as it has worked for my analyses of endolithic cyanobacteria): use (Ultra Clean Microbial DNA Isolation Kit, MoBio) (or equivalent kit for fungi, if t is available). Put one fungi sample in 150 µl Micro Bead Solution, destroy sample to small pieces with a dissecting needle, vortex for 30 sec. For cell-break, freeze-thaw samples in liquid nitrogen and 55°C hot water for five times (remaining 1,2 min at each temperature treatment). Afterwards add 50µl MD1 Solution (Ultra Clean Microbial DNA Isolation Kit, MoBio) and 60 µl Proteinase K (100 µg/ml) to the sample and keep at 50°C overnight. Proceed with the further steps of the used kit the next day.
Aref what kind of sample are you extracting? is mycelia in solid media? are dryed voucher specimens? sporocarps? mycelia in liquid media?
What fungi are you extracting? are their mycelia monomitic, dimitic or trimitic?
If your are extracting monomitic mycelia from fungi cultured in solid media the cell wall brakes easily with heat and some detergent so it usually is not the problem. Maybe you do have DNA but with inhibitors of PCR. Why you said it fails? because it do not amplify in PCR? have you run DNA gels or quantified it in a nanodrop?
dear Roberto
in fact, this sample have to be identified ... it's an unknown fungi which has contaminated students samples and we want to extract its DNA to identify the nature of fungi. it is mycelia in solid medium
thank you
here are two microscopic photos taken by a friend of mine from the fungi which we are about to take its total DNA
I have had a lot of success with Zymo fungal/bacterial kit.
https://www.zymoresearch.com/dna/microbial-environmental-dna-isolation-1/bacterial-fungal-dna/zr-fungal-bacterial-dna-miniprep-1
The very standard protocol is to get the fungal wall disrupted physically (mortal and pestle, Beads shocker, etc), treat with lysis buffer ( 200mMTris-HClpH7.5，0.5%w/v SDS，25mMEDTA, 250mM NaCl）, and, subsequently, phenol chloroform extraction. This should work whatever your fungus is.
This is very easy , you can follow the company protocol of Kit DNA, you can extract DNA on soild media like WA or liquide media BDA
Thank you
Sincerely
Aref, for aerial mycelia grown on petri dish the easiest way to extract DNA for PCR and sequencing a the kit XNAP of Sigma. It is a two step protocol, you just take a 1mm piece of mycelia in a PCR tube, put a 20 ul drop of extraction solution, then heat it 10 min at 65º, then 10 min at 95º, put a 20ul drop of dilution solution, 30 min at room temp are you are done¡ easy, chip, reliable, no chance for error.
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