OmniPrep™ For High Quality Genomic DNA Extraction From Yeast
Aliquot 1.5ml yeast overnight culture into a 1.5ml microfuge tube and centrifuge at 14,000xg for 30 seconds .
Discard the supernatant .
Add 150µl PBS , 5µl LongLife™ Zymolyase® and 1µl β - mercaptoethanol to the pellet and gently vortex to resuspend .
Incubate at 37°C for 30 minutes with periodic mixing .
Centrifuge for 5 minutes at 14,000xg and pour off the supernatant .
Gently vortex the tube to resuspend the pellet in the residual liquid .
Add 500µl Genomic Lysis Buffer and mix by inverting the tube several times .
Do not vortex .
Incubate the sample at 55 - 60°C for 15 minutes .
Do not heat higher than 60°C .
OPTIONAL : For maximum DNA recovery , 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 .
This step will digest hard to handle tissues and significantly improve the yield .
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 .
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 .
OPTIONAL : For increased DNA recovery , add 2µl Mussel Glycogen as a DNA carrier .
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 .
Add 50µl TE Buffer to the pellet .
Incubate at room temperature for at least 15 minutes to rehydrate .
OPTIONAL : 1µl LongLife™ RNase for every 100µl TE Buffer can be added at this stage .
Store DNA at 4°C , for long term storage store at - 20°C or - 80°C .
