Check this

http://www.biotechniques.com/biotechniques/multimedia/archive/00010/03355bm05_10134a.pdf

Is hard to extract DNA from fung due to tick cell wall.. So far more than most bacteria. The easy way is to use ultrasound to destroy wall. Should not be long to avoid the fragmentation of DNA. Another way is to use glass beads to destroy cell wall (in eppendorf tube)

Good Job

Dear Mohammad,

The best suggested method is as follows:

1-Inoculated aseptically the culture of  actinomycetes into 250 mL Erlenmeyers  each containing 30 mL of the respective media (YMG, MEM)

2- Incubate up to mid log phase in a rotary shaker at 30°C with 180 rpm.

3-Add glycine (20%) and MgCl2 into the media.

4- centrifuge the culture at 10,000 rpm for 10 minutes.

5-Wash the culture twice with lysis buffer.

6- Transfer the mycelia  into fresh tube containing 500 μl of TE buffer supplemented with lysozyme (50 mg/mL).

7-Incubate the mixture for one hour.

8-Take 100ul of this mixture and use Plant DNA isolation kit (Qiagen) for DNA isolation according to the manufacturer's instruction.

Another improved method can be found in the attached file:

Journal of Sci. Engg. & Tech. Mgt. Vol 2 (2), July 2010

An Improved Method for Isolation of Genomic DNA from Filamentous

Actinomycetes

1Vijay Kumar, 2Alpana Bharti, 3Omprakash Gusain, 4*Gajraj Singh Bisht

Abstract

A simple and efficient method involving the use of liquid nitrogen for rapid isolation of high molecular weight (≥ 50 kb) DNA from actinomycetes is described here. The DNA yields range from 140 to148.3 μg per g (wet weight) of mycelia with purity measured at A260/280 of 1.78-1.93. The DNA preparation is suitable for PCR protocols.

Hoping this will be helpful,

Rafik

Dear Mohammad,

The best suggested method is as follows:

1-Inoculated aseptically the culture of  actinomycetes into 250 mL Erlenmeyers  each containing 30 mL of the respective media (YMG, MEM)

2- Incubate up to mid log phase in a rotary shaker at 30°C with 180 rpm.

3-Add glycine (20%) and MgCl2 into the media.

4- centrifuge the culture at 10,000 rpm for 10 minutes.

5-Wash the culture twice with lysis buffer.

6- Transfer the mycelia  into fresh tube containing 500 μl of TE buffer supplemented with lysozyme (50 mg/mL).

7-Incubate the mixture for one hour.

8-Take 100ul of this mixture and use Plant DNA isolation kit (Qiagen) for DNA isolation according to the manufacturer's instruction.

Another improved method can be found in the attached file:

Journal of Sci. Engg. & Tech. Mgt. Vol 2 (2), July 2010

An Improved Method for Isolation of Genomic DNA from Filamentous

Actinomycetes

1Vijay Kumar, 2Alpana Bharti, 3Omprakash Gusain, 4*Gajraj Singh Bisht

Abstract

A simple and efficient method involving the use of liquid nitrogen for rapid isolation of high molecular weight (≥ 50 kb) DNA from actinomycetes is described here. The DNA yields range from 140 to148.3 μg per g (wet weight) of mycelia with purity measured at A260/280 of 1.78-1.93. The DNA preparation is suitable for PCR protocols.

Hoping this will be helpful,

Rafik

After apply one technique you can see the efficiency by using simple microscopy, you can see if the wall is broken. The high pressure will be new economic and powerful method for genomic extraction. Last time Gaston Hui Bon Hoa from national institute for health present this topic. I think the genomic extraction technique research has high impact in science. Application of simple method is priority, since the modern technique sometime interfere with genome integrity.

Check this protocol

1. Transfer 1.5 ml of the actinomycetes culture (grown in GYM medium for five days) to a 1.5 ml Eppendorf tube and centrifuge at max speed for 1min to pellet the cells.

2. Discard the supernatant.

Note: Remove as much of the supernatant as you can without disturbing the cell pellet.

3. Resuspend the cell pellet in 600 μl lysis buffer and vortex to completely resuspend cell pellet.

4. Incubate 1 h at 37 °C.

5. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.

Note: Do not vertex the tube—it can shear the DNA.

6. CAUTION: Phenol is a very strong acid that causes severe burns. Chloroform is a carcinogen. Wear gloves, goggles and lab coat, and keep tubes capped tightly. To be safe, work in the hood if possible.

7. Spin at max speed for 5 min at RT (all spins are performed at RT, unless indicated otherwise). There is a white layer (protein layer) in the aqueous: phenol/chloroform interface.

8. Carefully transfer the upper aqueous phase to a new tube by using 1 ml pipetman (to avoid sucking the interface, use 1 ml tip with wider mouth-cut 1 ml tip-mouth about ~2 mm shorter).

9. Steps 4-6 can be repeated until the white protein layer disappears.

10. To remove phenol, add an equal volume of chloroform to the aqueous layer. Again, mix well by inverting the tube.

11. Spin at max speed for 5 min.

12. Remove aqueous layer to new tube.

13. To precipitate the DNA, add 2.5 or 3 volumes of cold 200 proof ethanol (store ethanol at -20 °C freezer) and mix gently (DNA precipitation can be visible).

Note: DNA precipitation may simply diffuse, which is normal. Keep the tube at -20 degree for at least 30 min (the longer the better) and then spin it down (see Steps 15-16). You should see DNA pellet. It looks transparency when it is wet and turns to white when it becomes dry.

14. Incubate the tube at -20 °C for 30 min or more.

15. Spin at max speed for 15 min at 4 °C.

16. Discard the supernatant and rinse the DNA pellet with 1 ml 70% ethanol (stored at RT).

17. Spin at max speed for 2 min. Carefully discard the supernatant and air-dry the DNA pellet (tilt the tube a little bit on paper towel). To be faster, dry the tube at 37 °C incubator.

18. Resuspend DNA in TE buffer.

Note: Large amounts of RNA will be present in the DNA sample. So, for subsequent reactions, for example, to digest plasmid DNA, add 1-5 μl (1 mg ml-1) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to lysis buffer with a final concentration of 1 mg ml-1.

19. Check isolated Gemonic DNA on an agarose gel.

Note: We expect to see bands with smear patterns from high to low MW range, although most of DNA fragments are accumulated at high MW on the gel. So, if you see most of DNA fragments are small, very likely your DNA got degraded.

Source: Bio-protocol 2011 or http://www.bio-protocol.org/e97)