Method prepared by Grace Panganiban: gepangan@facstaff.wisc.edu


Hoffman-Winston Yeast DNA prep

1) Inoculate 5 mls (in labelled, sterile 15 ml culture tubes) of trp-glu+ liquid medium with a part of a yeast patch from the his-gal+ plates. Vortex to mix cells. Put on rotator in Culbertson lab (4th floor) 30oC incubator overnight.

2) Pour ~1.5 mls of culture into a labelled eppie; spin ~15 sec; pour off medium. Add another ~1.5 mls; spin; and pour off medium again.

3) Add 200 ul of Blue Buffer, 200 ul of phenol:chloroform, and 0.3 g (eppie scoop) of small glass beads to each pellet.

4) Vortex (using snazzy vortex attachment) for 3 minutes in 4th floor coldroom.

5) Spin for 2-3 minutes.

6) Recover ~180 ul of clear supernatant into clean, labelled eppie tube.

7) Add 500 ul of EtOH; mix; and spin for 5 min.

8) Remove EtOH with a pasteur pipette.

9) Add 1 ml of 70% EtOH; spin for 1 min.

10) Remove EtOH with a pasteur pipette.

11) Spin for 10 sec; remove remaining EtOH with a P200.

12) Resuspend pellets in 100 ul TE.

Blue Buffer
2% Triton X-100 10 mls 10%
1% SDS 2.5 mls 20%
100 mM NaCl 1 ml 5 M
10 mM Tris pH 8.0 0.5 ml 1 M
1 mM EDTA 100 ul 0.5 M
50 mls

Phenol:chloroform
25 mls TE saturated phenol, pH 8.0
24 mls chloroform
1 ml isoamyl alcohol

TE
10 mM Tris pH 8.0 0.5 mls 1 M
1 mM EDTA 100 ul 0.5 M
50 mls

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