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Isolation of Yeast DNA Prepare in advance

Summary: Isolation of Yeast DNA
Prepare in advance:
2.0 ml microfuge tubes containing 0.25 g of 0.5 mm acid washed glass beads and 250 l
phenol:chloroform:isoamyl alcohol at 25:24:1 (OH-CHCl3-IAA). Label and place on ice.
TENTS buffer: 100 mM NaCl, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 2% Triton X-100, 1% SDS.
Powdered dry ice (2-4 lbs).
1. Collect 5-10 OD units of yeast cells (e.g., 5 ml saturated SD culture at OD600 ~1, or 1 ml saturated
YPD culture at OD600 ~10). Centrifuge for 5 minutes at 2000 rpm. Discard supernatant.
2. Resuspend cell pellet in 0.5 ml TE buffer. Transfer to 1.5 ml microfuge tube. Spin at top speed for
1 minute. Aspirate supernatant. Pellets may be stored at -80C at this point.
3. Resuspend pellet in 250 l TENTS buffer. Do one tube at a time. Work quickly. Transfer to 2.0
ml tube with glass beads and OH-CHCl3-IAA. Tighten cap! Vortex 15 seconds. Place on ice.
4. Place tubes in multi-tube vortex mixer at 4C. Vortex for 30 minutes. Check cell breakage under
microscope; 50-75% is ideal. Vortexing too long will shear genomic DNA into small pieces.
5. Place tubes in powdered dry ice. Allow to freeze completely for 10 minutes.
6. Microfuge 10 minutes at top speed at room temperature. The tube will thaw during centrifugation.
Transfer supernatant (200-225 l) to a fresh 1.5 ml tube. Avoid transferring interface material.
7. Add 250 l of OH:CHCl3:IAA. Vortex 5 minutes in multi-tube vortex mixer. Spin 5 minutes at
room temperature. Transfer supernatant a fresh 1.5 ml tube. Avoid transferring interface material.
8. Measure volume. Add 2 volumes of 100% EtOH. Vortex. Hold on ice for ~5 minutes.


Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida


Collections: Biology and Medicine