Hi Rajender: I have use Extract-N-Amp™ Plant PCR Kit for DNA extraction from Fungi (Fusarium) and it work , Its simple and easy with one step of extraction, time for extraction is about 15 min,

This kit is for DNA extraction from plant tissue but is very useful for fungi.

Thanks you Mantny for suggesting  kit

Hi Rajender.

What do you want to do with your DNA?

I have used this CTAB method succesfully several times ,the trick to have clean mycelium is to put a layer of autoclaved cellophane (real, transparent cellulose sheet) betweenthat agar medium and the inoculum and then peel it of after a few days.

Method according to : Möller, EM, Bahnweg, G, Sandermann H and Geiger HH, (1992) A simple and efficient protocol for isolation of high molecular weigth DNA from filamentous fungi, fruit bodies and infected plant tissues. Nuc. Acids Res. 20-22 p 6115-6116.

Materials and solutions in eppendorf tubes

- 12 ml tubes - Chloroform/Isoamylalcohol (24/1)

- liquid nitrogen - 7.5 M NH4Ac

- TES: 100 mM Tris, pH 8.0 - 2-propanol

10 mM EDTA - 70 % ethanol

2 % SDS - 96 % ethanol

- 10 μg/l proteinase K - RNAse 10 mg/ml

- 5 M NaCl - T10E0.1

- 10 % CTAB

Method

- Or 100 l infected fruit tissue

- Grow mycelium on MEYAA or PDA plates with cellophane, peel of mycelium

- Freeze-dry O/N (optional)

- Grind the mycelium with glass balls OR a disposable pestle in TES (piece of 2x3 cm)

- Add 550 l of preheated TES and 10 μl proteinase K (10 /), vortex once and short to resuspend the powdered mycelium (+/- 50 mg)

- Transfer the tube to a 60C, incubate for 1 hour with occasional shaking.

- Add 140 l 5M NaCl and 65 l 10% CTAB

- Incubate 10’at 65C

- Add 700 l Chloroform/IAA and incubate on ice for 30’

- Spin 10’ at max at 4C

- Carefully pipet the aqueous phase (600 l)

- Add 225 μl 7.5 M NH4Ac and incubate at least 30 min on ice

- Spin 10’ at max at 4C and transfer to new tube

- Add 0.55 l isopropanol to precipitate DNA

- Spin 10 min at max, discard supernatant

- Wash with 70% ethanol and dry

- Dissolve in 0.2 ml TE containing RNAseA, incubate at 37C for 20’

optional- Precipitate with 1/10th V NaAc and 2.5 V 96% ethanol

- Wash and take up in 200 l TE

- Dissolve by incubating at 4C

Use 20-25 ng of gDNA for PCRs

In 10 l with 0.25 units of Taq

2.5 mM MgCl2, 200 M of each dNTP, 0.2 M of each primer (= 2 pmol), mix common primer with all 3 species specific in one tube.

- 95 C 2 min

- 35 cycles 95 C 15 sec

60 C 15 sec

72 C 1 min

- 72 C 3 min

- 15 C

hI Henk-jan Schoonbeek, 

I am using the same protocol for Botrytis fabae, I need a really pure DNA for sequencing so I am a bit struggling.

Anyway I was wondering which kind of cellophane membrane are you using? ( brand code).  From the one I use I can't peel off any mycelium ..

Thanks

Ciao Rosa.

Sorry I only just saw this. I really don't know brand names, I used what was in the labs already. It has to be REAL cellophane, some companies call any clear foodwrap cellophane. You know it is real when you press it with moist fingers for a while they will stick to it. The best search may actually be for jam covers. CUt them toi size and sterilise (wither moist, each between a layer of filter paper or many together shwimming in a wide brimmed beaker/pot/container. Take out with 2 sterile tweezers or they fold double. ALso grow you fungus as short as possible or it will grow through. I hope this still helps.

Henk-jan

Hi Henk - jan Schoonbeek and Rosa Caiazzo,

I am trying to get pure DNA from B. cinerea and fabae and I am really struggling on this. Can you give me the details of the protocol using autoclaved cellophane with growth condition? Have you get success in other media? If, please let me know which one?

Thanks.