High quality DNA extraction from Fungi_small scale
Collect tissue/spores (10-20mg) in a 2ml eppendorf tube with one metal bead.
lyse tissue/spores with liquid nitrogen in a precooled tissuelycer.
using 50Hz for 1min.
Make lysis buffer by mixing buffer A+B+C (2.5:2.5:1 + 0.1%PVP final) and briefly heat to 64 °C.
Let cool to room temperature, add RNAse T1 to lysis buffer according to 1:1750 of RNAse T1: lysis buffer ratio.
Add 500ul lysis buffer into the powder, briefly vortex, incubate at 64oC for 30 mins Cool on ice for 5 mins.
Add 100 ul (0.2 vol) of KAc 5M, mix by inversion, incubate on ice for max 5 mins.
Spin at 4oC at max speed for 10 mins.
Transfer supernatant to fresh eppendorf tube containing 500ul (1vol) (P/C/I) and mix by inversion for 2 mins.
Spin at 4 °C and max speed for 10 mins.
Transfer supernatant (~400ul) to fresh eppendorf tube containing 500ul RT isopropanol.
Incubate at RT for 5-10mins.
Spin at 4 °C and max speed for 30 mins.
Carefully remove supernatant with pipette and wash with 1mL fresh 70% Ethanol, invert several times to dislodge pellet.
Spin in table top centrifuge for 5mins at 13000g.
Remove supernatant with pipette and wash with 1mL fresh 70% Ethanol, invert several times to dislodge pellet.
Spin in table top centrifuge for 5mins at 13000g.
Remove supernatant with pipette.
Spin in table top centrifuge for 1min at 13000g.
Remove the remaining ethanol with pipette.
Air-dry pellet for 7mins.
Add 50ul of TE buffer and flick the tube slightly for mixing.
DO NOT vortex as it shears DNA.Store at -20oC.
