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 .
