protocol_21.txt

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.