Yeast Miniprep (Plasmid Rescue)
from Robzyk and Kassir, Nucleic Acid Res. (1992) 20:3790
Materials:
• 5 ml overnight culture yeast in drop out media (must be very dense)
• STET (8% sucrose, 50 mM Tris pH 8, 50 mM EDTA, 5% Triton X-100)
• Glass beads
• 7.5 M NH4OAc
• ice cold EtOH
• 70% EtOH
Procedure:
1) Spin down yeast culture in purple screw cap tube. Aspirate off liquid and resuspend in 100 µl STET.
2) Add 2 scoops of glass beads (approx 200 mg) and vortex for 5 min.
3) Add another 100 µl STET. Vortex briefly to resuspend.
4) Vortex for 5min (8min if using multihead vortex) at 4 degrees
5) Place tubes in 100degC heat block for 3 min. (cover tubes with weight). Alternatively, use boiling water bath for this step
6) Cool briefly on ice <1-2 min) and then transfer as much liquid as possible to a clean 1.5ml eppendorf using a blue tip (discard the beads).
7) Spin in microfuge for 10 min at 4degC.
8) Transfer 100 µl of supe to new tube.
7) Add 500 µl 7.5 M NH4OAc. Incubate at -20degC for 1 hr. or o/n
8) Spin 10 min at 4degC. (This step gets rid of impurities that inhibit transformations - should see small white pellet).
9) Collect 100 µl supe and add to 200 µl ice cold EtOH.
10) Spin 10 min at 4degC.
11) Discard sup and rinse pellet with 70% EtOH.
12) Centrifuge 5min, remove sup., dry pellet in speed vac
12) Resuspend pellet in 20 µl of water. Use 10 µl to transform bacteria. Can do a regular transformation.
