OmniPrep™ for Fungus
To avoid repeated freezing‐thaw , dispense the Proteinase K solution into aliquots of 30µl / tube and freeze at ‐20°C .
If a precipitate forms due to cold storage allow to warm to room temperature until precipitate dissolves .
Centrifuge the Molecular Grinding Resin tube for 2 minutes at 2,500x g and remove the water .
Add 0.5ml Genomic Lysis Buffer .
Collect fungal tissue from liquid culture and wash 2‐3 times in sterile water .
Fungal mycelia are best prepared by grinding samples using Molecular GrindingResin™ in Genomic Lysis Buffer .
Add 10‐20mg fungal mycelia to a microcentrifuge tube containing 500µl Genomic Lysis Buffer .
Resuspend Molecular Grinding Resin by vigorous mixing or vortexing .
Add 30µl Molecular Grinding Resin™ using a wide bore pipette tips and grind with a microcentrifuge pestle .
Add 1µl Proteinase K solution for every 100µl Lysis Buffer and incubate at 60°C for 1‐2 hours .
Invert the tube periodically each hour .
Allow the sample to cool to room temperature .
Add 200µl chloroform and mix by inverting the tube several times .
Centrifuge for 10 minutes at 14,000xg and carefully remove the upper phase to a clean microcentrifuge tube .
Add 50µl DNA Stripping Solution to the sample and invert several times to mix .
Incubate the sample for 5‐10 minutes at 60°C .
Add 100µl Precipitation Solution and mix by inverting the tube several times .
A white precipitate should be produced , if not add 50µl aliquots of Precipitation Solution until a white precipitate forms .
Centrifuge the sample at 14,000xg for 5 minutes .
Transfer the supernatant to a clean tube and precipitate the genomic DNA with 500µl isopropanol .
Invert the tubes 10 times to precipitate the DNA .
Centrifuge at 14,000xg for 5 minutes to pellet genomic DNA .
Remove the supernatant .
Add 700µl 70 % ethanol to the tube and invert several times to wash the DNA pellet .
Centrifuge for 1 minute at 14,000xg .
Decant or pipette off the ethanol wash .
Invert the tube on a clean absorbent surface for several minutes to allow any excess ethanol to drain away .
Do not let the pellet dry completely or it will be difficult to rehydrate .
Add 50 to 100µl TE Buffer to the pellet .
Incubate at room temperature for at least 15 minutes to rehydrate .
Incubating the tube at 55‐60°C will speed up rehydration .
Incubate for 5‐60 minutes .
OPTIONAL : Add 1µl LongLife™ RNase for every 100µl TE Buffer at this stage .
Store DNA at 4°C , for long‐term storage store at ‐20°C or ‐80°C .
