Yeast RPM Kit
Revision No. 2069-999-5F03W

This is a dual purpose kit for the isolation of plasmid DNA from yeast and bacteria and is especially useful during screening studies of two-hybrid transformants.

1. Transfer 1.5 ml of yeast culture to a microcentrifuge tube; spin for 30 seconds to pellet the cells and decant the supernatant.



2. Add 250 µl Alkaline Lysis Solution and ~0.4 g Yeast Lysis Matrix (0.4 g occupies a volume corresponding to 250 µl). Vortex continuously for 5 minutes at room temperature or shake 10 seconds in a FastPrep Instrument.




Note: The presence of detergents in the Lysis Solution will cause the sample to foam. To facilitate the processing of multiple samples, use a multi-tube holder attached to the vortex machine. FastPrep is a controlled high-speed shaking device available through BIO 101 to extract nucleic acids from virtually any source: cells, tissues, organs, and organisms; it can process up to 12 samples simultaneously (see pg 70).




3. Add 250 µl Neutralizing Solution; mix by brief vortexing and spin 2 minutes at room temperature. Transfer the supernate to a Spin Filter, avoiding the precipitated debris and Lysis Matrix.



4. Add 250 µl Glassmilk Spin Buffer; invert to mix; spin 1 minute and empty the Catch Tube.



5. Add 500 µl Wash Solution; spin 1 minute and decant the wash from the Catch Tube. Repeat the wash step; decant the Catch Tube and spin for 1 minute to drive the last of the liquid out of the Spin Filter. Transfer the Filter to a new Catch Tube.



6. Add 100 µl sterile H₂O; vortex briefly (at no more than half speed) to resuspend and spin 30 seconds to collect the DNA in the bottom of the Catch Tube; discard the Filter containing used Matrix. Use 5 µl for transformation and PCR analysis.