Quick Fungal DNA Extraction
Inoculate fungal material into 300 µL rich growth medium in a 2 mL micro - centrifuge tube . Grow at RT for 3 - 4 days .
Freeze culture tubes in LN2Freeze dry over night .
Add 50 µL 0.6mm glass beads to freeze - dried sample in 2 mL tube and grind using Tissue - Lyser LT for 5 mins at 50 Hz .
Add 500 µL lysis buffer to pulverised sample .
Vortex to mix Heat to 45 ˚C for 5 mins , shaking at 1200 rpm .
Add 0.5 vol ( 250 µL ) 3 M Potassium Acetate to lysis mix .
Vortex to mix .
Centrifuge at 12 K rpm for a minimum of 15 minutes @ 4˚C .
Transfer supernatant ( ~ 650 µL ) to fresh 1.5 mL microcentrifuge tube .
Add 1 vol chilled Isopropanol ( ~ 700 µL ) to the supernatant , mix well , incubate - 20˚C for 1hr .
Centrifuge at 12 K rpm for a minimum of 15 minutes at 4°C .
Decant gently or aspirate supernatant .
To the pellet add 500 µL 70 % ethanol stored at - 20°C .
Centrifuge at 12 K rpm for a minimum of 3 minutes at 4°C .
Discard supernatant .
Repeat wash step with 500 µL of cold 70 % ethanol .
Spin and discard supernatant .
Dry pellet in speed - vac at 45˚C for 5 - 10 minutes .
Re - suspend pellet in nuclease free water or TE buffer ( 10 mM Tris pH 8.0 , 1 mM EDTA pH 8.0 ) .
Add 50 µL 3M Sodium Acetate pH 5.5
