If you are isolating fungal DNA from soil, then you can use PowerSoil DNA isolation kit. But if you are isolating from fungi culture, then, you can try this https://pdfs.semanticscholar.org/c8ef/4d8b01b90b9bfd1b2879e03e776d7cd3f0fc.pdf . If you don't have liquid nitrogen , you can use homogenizer instead

Isolation of fungal from soil:

Isolated the fungal from the collected soil sample by serial dilution method, each sample 4 dilution 10-1 to 10-4.

Prepare the 250 ml of Sabouraud dextrose broth sterilised by autoclaving.

Transfer the 10 ml of Sabouraud dextrose broth to each tube.

Weight about 1gram of each soil sample and add to the first tube 10-1.

From that take 1ml and transfer to next tube, continue the process till the last dilution.

Prepare 500ml of potato dextrose agar

The plating was done by the method called pour plat, here take 1ml of dilution transfer to sterile petriplate and add the potato dextrose agar mix it slowly and properly. Incubate the plates at room temperature under light and under dark for 3 days. Observe the plates daily and note down the observation.

After 3 days store the plates under 40 c.

The plates are contaminated with some of bacterial colonies so again inoculate the fungal in new potato dextrose agar plates. Cut the fungal part from the plate carefully without touching to bacterial colony and placed on the new plate and again incubate at room temperature for 3 days.

Inoculate the fungal for the DNA isolation

Prepare Sabouraud dextrose broth sterilised by autoclaving in a 100ml flask.

Approx 50 ml of Sabouraud dextrose broth for and inoculate the small colony and incubate under room temperature for 3days in shaker incubator.

On 4th day in a sterile condition under “Laminar air flow” filter the broth along with the fungal mycelium mat through a filter paper and dry the sample. The dried fungal mycelium mat was transferred to sterile ependoff tube and store under -80oc .

Fungal DNA isolation buffer:

DNA ISOLATION:

v 0.5M EDTA (EthyleneDiamine Tetra Acetic Acid)

v 1M Tris (pH 8).

v 10% SDS (Sodium Dodecyl Sulphate).

v DNA entraction buffer (STE buffer).

v Tris saturated phenols.

v Chloroform:Isoamylalcohol (48:2).

v Isopropanol.

v 70% Ethanol.

v Sterile Milli Q water.

v 6x Gel loading dye.

v Agarose.

v Ethidium bromide (EtBr 10mg/ml).

v 10X TE Buffer.

v 1X TE Buffer.

Sodium Tris EDTA (STE) extraction buffer:

1M Tris

o.5M EDTA

5M NACL

10% SDS

Sterile milli Q water

Sterilised by autoclaving 121oC for 15 minutes at 15 lbs pressure.

Procedure:

Ø Approximately 100 mg of fresh mycelium was placed into 1.5 ml of micro centrifuge tube. To this add 750 µl of extraction buffer, 50 mg of glass powder, 50 mg of poly vinyl poly pyrolidene(pvpp) were added.

Ø The mucelium was grind using plastic mini grinder (sigma) and the tubes were incubate at 65o c for 15 min and then centrifuge at 10000 rpm for 10 min.

Ø Transfer the supernatant to new eppendoff tube and added 5µl of RNase (10mg/ml) and incubated 37oc for 20 minutes.

Ø After incubation added equal volume of phenol: chloroform: isoamylalcohal (25:24:1) mix gently and centrifuge 12000 rpm for 15min at 4oc.

Ø Transfer the aqueous layer to new strile eppendroff tube and added equal amount of chloroform: isoamylalcohal (24:1), mix gently and centrifuge at 12000rpm for 15min at 4oc.

Ø Transfer the aqueous laye to new ependoff tube and added 2/3 volume of isoproponal and incubated at room temperature for 10 minutes to precipitate DNA and centrifuge 12000rpm for 15 minutes.

Ø Pour the solution carefully and to the pellet added 200µl of ice cold 70% ethanol, centrifuge at 10000rpm for 5 minutes at 4oc.

Ø The pellet was air dried and resuspend in 50µl of sterile milli Q water.

Ø The DNA were store at -20oc.

thanks to everyone for your answers, i'll let you know soon about my progress in the matter

Hi again, i'll provide you with a bit of context:

Right now I'm preparing a diverse types of nutritious broths for the fungi (including Sabouraud, malt extract, potato dextrose). The thing is that i recently discovered that this fungi has an obligate endohyphal bacteria, and maybe sabouraud (acidic broth) will prevent the growth of endohyphal bacteria, and i don't want to do metagenomics yet.

Fungal DNA isolation here (in our lab) it's a bit complicated because liquid nitrogen always came with a sort of contamination and always we get a bad cell wall lysis. I already did the classic 16s and the results were pretty weird, nanodrop show us that the DNA was horrible contaminated (probably due the bad cell wall lysis), and for now this is what i have.

So, for now I will follow your suggestions and update this thread as soon as possible.

Thanks for your answer, sorry for grammar mistakes.

1. This fungus

2. An obligate endohyphal bacterium

3. That's a pretty nice information, i'll take your suggestions.

I'll let you know soon about my progress.

Regards

ahh ok, sorry for that. I made the correction.