The right answer will vary greatly with the group of fungi and type of sample you have. Not all fungal extractions require the use of liquid nitrogen as other means of mechanical lysis can be explored (bead beating, bead beating with abrupt changes in temperature, or a micro-tube pestle might work for fresh tissue samples).

For extraction from fresh samples, you might want to follow the methods on this paper:

Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis) in Peru

Romina GAZIS*, Priscila CHAVERRI

For extraction from soil, follow this one:

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics􏰉†

Marketa Sagova-Mareckova,1* Ladislav Cermak,1 Jitka Novotna,1 Kamila Plhackova,1 Jana Forstova,2 and Jan Kopecky1

If you still want to just do direct PCR, you could look at this option:

http://www.clontech.com/US/Support/Applications/PCR/Direct_PCR

Hope it helps,

Happy Research!

Check this http://www.norgenbiotek.com/display-product.php?ID=54

Direct Fungi DNA Isolation Kit

(Cat# 25600)

For the rapid and convenient isolation of fungal DNA from plant surfaces

Rapid isolation of fungal DNA from plant surfaces

No liquid nitrogen or physical disruption methods required

Isolate high quality DNA for a wide range of downstream applications

Use the direct fungal isolation kit from Norgen. It give a pure and high through put analysis of the fungal DNA of your interest.

Fungi have a robust cell wall which requires mechanical (glass beads), chemical (Lyticase) or other methods for disintegration in order to release the DNA from the cells or spores. Conventional chemical lysis kits, heat shock or just add tiny sample into the PCR mix does not work, also I have no experience with liquid nitrogen but do not think it is useful as spores are shown to survive even "space conditions".

No kit was used in this paper but a modification of the HotSHOT method by Truett (original reference cited therein). You can try this approach.

Depends of what kind of fungal tissues are you starting from (i.e., mycelia, spores, fruit bodies, ectomycorrhizas) and how it was served (i.g., fresh, dried, frozen, lyophilized)... I use REDExtract-N-Amp™ Plant PCR Kit with great results.

http://www.sigmaaldrich.com/catalog/product/sigma/xnap?lang=it®ion=IT

You might want to try a simple 'Chelex' quick prep method for fungal mycelium as described in this publication:

Wirsel, S. G. R., Runge-Froböse, C., Ahren, D. G., Kemen, E., Oliver, R. P., Mendgen, K. W. 2002: Four or more species of Cladosporium sympatrically

colonize Phragmites australis. Fungal Genetics and Biology 35, 99–113.

It also works without liquid nitrogen (only mechanical disruption), and yields crude extracts that are suitable for most downstream applications.

As long as you need just DNA from a fungi sample for research use several techniques or kits may work satisfying. But if you need to detect few fungi cells/spores in up to 10 ml human whole blood for approveal / disapproval of e.g. "invasive aspergillosis" in transplant patients of critical condition you may need clinical validated diagnostics extraction kits like the QIAamp UCP PurePathogen Blood Kit. A downstream ready-to-use PCR kit will be available soon. A clinical study using the kits is described in "Springer et al. 2012, 50(8), 2585-2591.

Did anyone try Chelex with prior incubation with Lyticase? I wonder if Lyticase could be used instead of mechanical disintegration in fungi other than yeasts as well.

The right answer will vary greatly with the group of fungi and type of sample you have. Not all fungal extractions require the use of liquid nitrogen as other means of mechanical lysis can be explored (bead beating, bead beating with abrupt changes in temperature, or a micro-tube pestle might work for fresh tissue samples).

For extraction from fresh samples, you might want to follow the methods on this paper:

Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis) in Peru

Romina GAZIS*, Priscila CHAVERRI

For extraction from soil, follow this one:

Innovative Methods for Soil DNA Purification Tested in Soils with Widely Differing Characteristics􏰉†

Marketa Sagova-Mareckova,1* Ladislav Cermak,1 Jitka Novotna,1 Kamila Plhackova,1 Jana Forstova,2 and Jan Kopecky1

If you still want to just do direct PCR, you could look at this option:

http://www.clontech.com/US/Support/Applications/PCR/Direct_PCR

Hope it helps,

Happy Research!

I've used Sigma extract-n-amp-plant, for Ustilago maydis sporidia (yeast-like cells) I never used on mycelium, so I don't know if it works or not.

Thank you so much friends. All time I am working with pure and fresh culture so I will go for Norgen kit.

Dear, Youssuf Sir, I think no Kit require here, just take your culture and make a suspension with st water with in a tube, kept the tube into a boiling water bath for 10 min and u directly use this suspension to PCR reaction tube with changes initial duration of deneturation temperature to 5-6 min (95 for 5 min). hope you will success with sufficient amount of amplicons. Thank you.

For Silke,

I am wondering the same question. May enzymes make the "dirty job" of cell degradation before DNA extraction? It surely could represent a valid option.

G.

For Gian and Silke,

I have routinely done this with fungi when I need to isolate large unsheared genomic DNA. Essentially you make protoplasts out of the mycelia. Wash these in an osmotic buffer and then lyse them using a detergent. I use CTAB. This works provided that you can make the protoplasts. There are several enzyme mixtures that work well for this. Most of them are sold as additives for wine making such as Novozymes Vino Taste Pro.

Thank you so much, Kenneth and Gian. This information might help to further improve my DNA extractions from lichen tissue.