Hello,
I am trying to extract DNA from different species of fungi but till now no luck (i am getting like 3ng/ul). I am using frozen in -20°C fresh mycelium collected from solid PDA medium. The main problem i think i am having is with cell lysis: I tried few things:
1. Grinding mycelium in Liquid Nitrogen within 1,5 ml tube with micropestle and then go on with further steps in Qiagen Manual.
2. Freeze/thaw method: I added 400 ul of AP1 to PCR tube with mycelium , vortex and then 7 times in cycle: 1. put a tube in liquid nitrogen until is completely frozen, then thaw on thermoblock at 80°C for a while - until buffer liquefies, put back in liquid nitrogen, and after repeats grind reaming clumps of mycelium with micropestle.
3.Freeze/thaw method + Boiling method - Exact like in method 2. but with additional step - boiling at 85°C for 0,5h and then go on with Qiagen manual.
Am i doing something wrong ? Methods which i tried was inspired by Griffin et al. 2002 Article A rapid and efficient assay for extracting DNA from fungi
andArticle A simple method of genomic DNA extraction suitable for analy...
Should i change a lysis buffer ? or maybe invest in tissue homegenizer with glass/zirconium beads ?
Hello Michal,
forget about freezing and grinding. Some fungi like Aspergillus become as sticky as chewing gum.
Mathew is right. Using silica beads in 2ml lysis tubes works excellent. But in order to do this efficiently you need a bit of equipment: you need a mill to shake the tubes vigourously (5000rpm/min). You can centrifuge the tubes for 2min at 10.000rpm and use the supernatant for your DNA extraction. The best way to do this would be to use a nucleic acid extraction platform.
You can also use phenol-chloroform. The most important thing is to disrupt your fungus.
Best regards
Dirk
Hi
It is better to culture each fungus isolate in PDB ( Potato Dextrose Broth) and incubate them on a shaker for about a week. Then you can use the formed colony of fungus for DNA extraction stages. It is increased quality and quantity of extracted DNA.
I suggest you use CTAB buffer.
Good luck
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