THERE'S A GREAT METHOD IM USING IT FOR FUNGAL DNA EXTRACTION

To a 1.5-mL Eppendorf tube containing 500 mL of lysis buffer (400 mM Tris-HCl [pH 8.0], 60 mM ethylene diaminetetra acetic acid [EDTA] [pH 8.0], 150 mM NaCl, 1% sodium dodecyl sulfate), a small lump of mycelia from young culture is added by using a sterile toothpick, with which the lump of mycelia is disrupted. The tube is then left at room temperature for 10 min. After adding 150 mL of potassium acetate (pH 4.8; which is made of 60 mL of 5 M potassium acetate, 11.5 mL of glacial acetic acid, and 28.5 mL of distilled water), the tube is vortexed briefly and spun at 10000 × g for 1 min. The supernatant is transferred to another 1.5-mL Eppendorf tube and centrifuged again as described above. After transferring the supernatant to a new 1.5-mL Eppendorf tube, an equal

volume of isopropyl alcohol is added. The tube is mixed by inversion briefly. The tube is spun at 10000 × g for 2 min, and the supernatant is discarded. The resultant DNA pellet is washed in 300 mL of 70% ethanol. After the pellet is spun at 10000 rpm for 1 min, the supernatant is discarded. The DNA pellet is air dried and dissolved in 50 mL of deionized H2O, and 1 mL of the purified DNA is used in 25 to 50 mL of PCR mixture. The extractions were done in duplicate assays for each sample.

I recommend the DNeasy Plant Mini Kit (Qiagen), I found it very useful in extracting DNA from Aspergillus

To extraxt fungal genomic DNA I also use the GenElute Plant Genomic DNA Miniprep Kit (Sigma Aldrich) with great success.

you can use CTAB extraction method..

Http://jcm.asm.org/content/38/1/471.full

my doubt when DNA isolated, DNA obtained is Genomic DNA or plasmid DNA? is there any method to obtain plasmid DNA from fungi???

How about a protocol to extract DNA direct from frutification bodies?

but if there is resistance observed to any environmental stress in fungi....it may or may not b due presence of plasmid !!! Is tht correct?

A DNeasy Mini Plant extraction kit (Qiagen Inc., Valencia, California) should be

used to extract DNA directly from fructification bodies (mushrooms, ascomata, etc.) following the manufacturer’s protocols, except tissues may or may not be ground in liquid nitrogen. In my lab, ascomata are

initially rehydrated in 100 mL of the AP1 buffer for 3–5 h, followed by freezing for 1+ weeks at -80 C. The trick is to collect enough material (at least 20-30 ascomata if working with pyrenomycetes, loculoascomycetes, Eurotiales, etc.).

Zymo Research has the best kit for fungal DNA isolation.

cheers,

shenoy

DNA extraction kits are very reliable in this search. I've used the Qiagen kit of the Dneasy mini plant extraction and that of DNA Miniprep Kit (Sigma Aldrich) and they we both very efficent and suitable.

Dear Andre,

Fungal DNA isolations can sometimes be trickier than you initially think - especially if you have species that have a high polysaccharide content. I have tried a wide range of protocols and kits. The biggest problem I found with the Qiagen whole genome DNA isolation kit was that negatively charged sugar would preferably bind the membrane leading to low DNA quantities that are still contaminated. That is no problem if you want to use the DNA for easy molecular manipulations such as PCR but might be a little more tricky if you want to use the DNA for sequencing. I recommend the MasterPure Yeast DNA kit from Epicentre. I know it says yeast but I have successfully used it for filamentous Candida and think it might be worth giving it a shot for your Aspergillus.

Hope that helps a little!

Best,

Kerstin

Dear Kerstin,

Thx for this important infiormation, well you gave us a new way to isalte fungal DNA in case we face a problem in the extraction with other methods...however till now i didn't face any problem in the extrcation of fungal DNA coz i use many methods like the CTAB, the fast DNA extraction, etc...and i used the DNA for RT-PCR and i have a high correlations.

NB: The question of DNA extraction was asked by M. Akarpu Rajender Reddy and not by me :)

Dear colleagues maybe you know the relatively simple method for fungi DNA extraction from mammals tissues?

Quick DNA extraction for fungi

100-200mg plant tissue or a weft of mycelium ( may include agar media plug)

place in 1.5 ml eppendorf tube containing 100-150µl 0.5M NaOH

macerate quickly with micro-pestle ( no big chunks) and allow to stand for 6-10 min

add 500µl of 100mM Tris-HCl 0.5M EDTA pH8,(sterile), vortex

centrifuge for 10 min at 14,000 rpm

transfer supernatant to a new labeled eppendorf tube.

store at -20°C

1µl is usually sufficient for PCR

Quick DNA extraction for fungi

100-200mg plant tissue or a weft of mycelium ( may include agar media plug)

place in 1.5 ml eppendorf tube containing 100-150µl 0.5M NaOH

macerate quickly with micro-pestle ( no big chunks) and allow to stand for 6-10 min

add 500µl of 100mM Tris-HCl 0.5M EDTA pH8,(sterile), vortex

centrifuge for 10 min at 14,000 rpm

transfer supernatant to a new labeled eppendorf tube.

store at -20°C

1µl is usually sufficient for PCR

i followed Broda and Reader method.very useful

Fresh mycelium of F. moniliforme var. subglutinans were collected and used for the genomic DNA was extraction according to the protocol of Abd-Elsalam et al (2003). The fungal mat were homogenized using 300 µl of extraction buffer containing 200 mM Tris-HCl (pH 8.5), 250 mM NaCl, 25 mM EDTA and 0.5 % of sodium dodecyl sulphate for 5 min. Add 150 µl of Sodium acetate (pH 5.2) and cooled for 10 min at 20 ºC. Centrifuge at 13,000 rpm for 5 min, supernatant transferred in fresh tube and added the equal volume of isopropanol. Precipitated DNA was then subjected centrifugation at 13,000 rpm for 10 min collect pellet and washed with 70% ethanol two times for the removal of excess salt. Dry out the pellet containing DNA and resuspended in Tris-EDTA (10 mM) and stored at 4ºC. DNA was quantified using standard spectroscopic methods.

We review some solutions in Hyde et al. 2013

(http://www.creamjournal.org/PDFs/Cream_3_1_1.pdf)

Another relevant review article is http://onlinelibrary.wiley.com/doi/10.1111/nph.12243/full (open access)

Rolf H. N.

I appreciate the pdf recommended have been useful, as a guide to standardize our protocols.

Thanks

LIIBAAM Lab-University of Santander

I achieved an extraction in this order......... Isolates of pure fungal strains (from feed samples) for DNA extraction were sub-cultured on (YES) broth medium and incubated for 7 days at 25 oC. The extraction of DNA was performed using a DNA extraction Mini kit (Quaigen). Mycelia of a pure fungal strain (100 mg) were harvested by filtration into a collection microtube, disrupted for 1 min using a tissue disruptor and 400 μl of Qiagen buffer AP1 and 4 μl RNase A (100 µg/ml) were added. This was vortexed and incubated for 10 mins at 65 oC with tube inversion 2-3 times during incubation. Buffer AP2 (130 μl) was added and mix with further incubation for 5 mins on ice. The lysate was pipetted into a QIAshredder Mini spin column in a 2 ml collection tube and centrifuged for 2 mins at 20,000 x g (14, 000 rpm). The flow-through was transferred into a new tube without disturbing the pellet formed. One and half of buffer AP3/E was added and mixed by pipetting. The mixture (650 μl) was transferred into a DNeasy Mini spin column in a 2 ml collection tube and centrifuged for 1 min at 8000 rpm (6000 x g). The flow through was discarded, repeating the process with the remaining sample. The spin column was placed into a new 2 ml collection tube with the addition of 500 μl Qiagen buffer AW. This was centrifuged for 1 min at a revolution 8000 rpm and the flow-through was discarded. Again, 500 μl buffer AW was added and centrifuged for 2 mins at 14, 000 rpm. The spin column was removed from the collection tube carefully and was transferred into a new 2 ml micro-centrifuge tube. Qiagen buffer AE (100 μl) was used for elution of DNA, which was incubated for 5 mins at room temperature and centrifuged for 1 min at 8000 rpm. This step was repeated for ensure complete elution and purification. The purified DNA was stored at -20 0C until further analysis.

Thanks Henry, other one for us.

I agree with Maria, the Qiagen plant kit works great for me, although with some added modifications to maximize yield.

Digest the cell wall of fungi using chitinase , centrifuge the content collect the supernatent and go for DNA extraction

Yes, well be careful about mutagens in the growth media (Paterson and Lima 2013; 2014) as these could mutate your DNA...

I thought I would expand on my previous answer about mutagens in growth media. Fungi are often isolated from the environment on media that contain mutagenic antibiotics to control bacteria. Also, when fungi grow they often produce mutagenic secondary metabolites such as mycotoxins. These compounds could mutate the DNA of the fungus even before it is isolated. Media need to be designed that reduce these compounds. As mentioned previously, I published recently on these topics.

since the structure of fungi is similar with a plant cell, any type of plant DNA isolation method should be adaptable. Also extraction kits are available particularly in spin column format. It has advantages over many conventional methods (rapid, easy to use) however the price, sample lysis (liquid nitrogen vs bead beating, phenol vs non-phenol) and processing time have to be considered.

Fungal cells are different from plant cells, so be careful here. For example, fungi have chitin in cell walls. Plants can have lignin in cell walls (and much cellulose). Fungi have ergosterol in cell membrane. Plants have chloroplasts of course.

Hello Russell, Sorry for the confusion. I have to clarify that the similarity what I meant came from the cell well lysis approach that unlike other bacterial cell structure, it is rigid and requires a physical rupture to get more effective DNA isolation. In my experience with Aspergillus Niger (spore), I found that the combination (lyticase + bead beating) improved the DNA yield.

I suggest the following method:

Four discs (5 mm diameter) of single-spore isolates of your fungus will be inoculated into 250 ml flasks containing 50 ml of potato dextrose broth (PDB) medium. After 4-6 days of incubation on a rotary shaker (100 rpm and 20°C), mycelia will be harvested from the flasks by vacuum filtration using two layers of sterilized cheese cloth, lyophilized for 5 days and stored at -30°C. Approximately 50 mg of the lyophilized mycelium will be transferred to microfuge tubes and re-lyophilized for an additional day. Mycelium of each isolate will be ground to a fine powder using a bead beater. DNA will extracted using a modified mini-preparation protocol using the cetyltrimethylammonium bromide (CTAB) method (Chongo et al., 2004; Vail and Banniza, 2009; Atik et al., 2011). The quantity and quality were assessed by running 1µl of the DNA on 1% agarose gel,

Do you apply Liquid Nitrogen at begining step of DNA isolation? We are now facing problems. The liquid nitrogen makes dificulty grounding mycelium to powder, it is stack. Even mycelium was dried by soft tissue after picked up from liquid culturing. 

Any suggetions are highly appreciated.

we have found that bead beating with lysis buffer (any available or home made) was effective for any downstream applications such as qPCR and NGS. 

Hi

we developed a quick method (using microwave)  that works great for sequencing in Fusaria and should easily apply also to Aspergillus. if you wish to give a try you found it here

https://www.researchgate.net/publication/262603748_Validation_of_a_Quick_PCR_Method_Suitable_for_Direct_Sequencing_Identification_of_Fusarium_Fungal_Species_and_Chemotypes_for_Preventive_Approaches_in_Food_Safety

good luck!

Article Validation of a Quick PCR Method Suitable for Direct Sequenc...

 Hello Tran Ho Quang: If using a mortar and pestle, try to form a thin layer of mycelium and if it has sticking to the bottom of the mortar, unstick it before adding nitrogen (you can do with a spatula).

Hello, there are several methods discussed under this thread but I need to focus on factors affecting DNA degradation since my aim will be full genome sequencing. Brief procedure; I am using Bioneer exiprep automated system for DNA extraction. I first ground potato dextrose broth-grown and then dried Fusarium mycelia mass (~100 mg) in liquid nitrogen, then add lysis buffer and proteinase-K, then 120 min in water bath at 60°C with soft shaking, followed by a centrifuge at 16500 xg for 5min then use supernatant for extraction process. No resuspension buffer used.  The automated system uses magnetic beads method. Although resulting DNA is highly pure according to spectrometer, agarose gel run shows degradation. Looking for comments for possible reasons of this degradation, particularly among the steps I provided above. I will be grateful for the answers/comments.

Hello, the attached file contain a very good method to isolation of DNA from any kinds of fungi.

Niazmand has a good protocol. We use CTAB with Proteinase K or PVP all the time, because DNA can be extracted from almost any fungus. If you know which fungus you have in hand, some kits are usable. For identification and not knowing which fungus you work with an general extraction protocol is better. We don't use ß-mercaptoethanol.

I generally use the CTAB extraction method. Aspergillus is a dry-sporulating genus with hydrophobic conidia. Contact with the buffer and conidia is minimal and difficult to extract DNA from. Use young cultures and sample hyphae before the colony starts sporulating. Needless to say to prevent loosing the DNA pellet throughout the whole procedure. If you need to use the Qiagen kit you could start with some mechanical lysis with glass or zirkonium beads or macerate the tissue with liquid nitrogen.

This is impossible, although your story says otherwise. This (probably) means, that somewhere along the procedure something goes wrong; a. you loose your pellet, b. DNAses are still active, c. something is wrong with the nanodrop and maybe some other reasons. Did you try a PCR of the ITS region with your samples or run the sample on agarose gel to check the quality of the DNA? It also excludes possible  problems with the nanodrop. The kit or CTAB should give a reasonable amount of DNA. Be sure to obtain and keep the extraction buffer pH high, like 8-9. This ensures the DNA to stay in the soluble water phase. Only during pelleting the pH can be decreased. Your problem is puzzling me. It is difficult to solve a problem from a distance.

Hi, You can use CTAP method its very easy,

Hi Akarapu

This references very useful for you

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=2ahUKEwjh0pGM-q_fAhWCsaQKHTskASQQFjAAegQIChAB&url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC4909022%2F&usg=AOvVaw2E_0qon19oHxxRHk8OGig9

Second one

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=2ahUKEwjh0pGM-q_fAhWCsaQKHTskASQQFjABegQIAxAB&url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC88759%2F&usg=AOvVaw1bdxDt6M0nOFSIvdwQ05Bu

Third

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=rja&uact=8&ved=2ahUKEwjh0pGM-q_fAhWCsaQKHTskASQQFjADegQIBhAC&url=http%3A%2F%2Fwww.funpecrp.com.br%2Fgmr%2Fyear2010%2Fvol9-1%2Fpdf%2Fgmr680.pdf&usg=AOvVaw3XJgLnh5ohqw6boXM2iwzM

Fungi DNA Isolation Kit provides a rapid method for the isolation and purification of total DNA from a wide range of fungi species.