“Smash N Grab” (SnG; phenol chloroform Yeast Genomic DNA Prep) Edit

1. Grow small cultures (usually 3 ml) overnight at 30°C in YPD

2. Fill a 1.5ml microcentrifuge tube with the culture and collect the cells by 1 min centrifugation.

3. Aspirate the supernatant off and briefly vortex the tube to loosen the pellet in the residual liquid.

4. + 200 μl of SnG lysis buffer [2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris-Cl (pH 8), 1mM Na2-EDTA].

+ acid-washed glass beads

Vortex to resuspend pellet

+ 200 μl of phenol:chloroform:isoamyl alcohol (25:24:1) in the fume hood

Vortex for 30 sec.

4. Centrifuge for 10 minutes at top speed in a microcentrifuge.

5. Remove ~160 μl supernatant (top layer, do NOT collect lower layer or interface) to new 1.5ml tube

6. + 400 μl 100% ethanol

 Allow a 5 min incubation at room temp.

7. Centrifuge again for 5 min, top speed.

8. Discard most of supernatant (do not disturb pellet)

     + 500 μl 70% ethanol.

9. Spin down 3 min full speed

Remove as much ethanol as possible without disturbing the pellet (ok to leave some).

10. Dry OVERNIGHT (or in speed vac 10 mins).

AFTER PELET IS DRY (e.g., next day)- Resuspend in 50 μl H2O.

Discard waste appropriately (in waste containers in fume hood)

For 50ml of SnG Lysis buffer

    5  ml   10% SDS

  10 ml   10% Triton X-100

    1 ml     5 M NaCl

    1 ml  0.5 M 1 M Tris-Cl, pH 8.0

0.25 ml 0.2 M EDTA

Water to 50ml