Yeast Cell Lysis Preparation Kit
For Use Prior to DNA and RNA Isolation Protocols
Revision No. 2015-999-6A01W

The Yeast Cell Lysis Preparation Kit contains the necessary reagents for removal of the yeast cell wall in preparation for efficient lysis for use with the G NOME DNA Isolation Kit (see pg 58). The rapid spheroplasting method employed in this protocol is suitable for genomic and plasmid DNA isolation. For an appropriate spheroplast transformation protocol, see Yeast Spheroplast Transformation Kit pp. 258-259.

1. Start with 100 ml of yeast, grown to a density of 2 x 10⁷ cells/ml.
2. Pellet at 600 xg for 5 minutes.
3. Discard supernatant and resuspend pellet in 1 ml of Yeast Suspension Buffer. Transfer to a microcentrifuge tube and centrifuge for 10 seconds.
4. Discard supernatant and resuspend in 500 µl of Yeast Enzyme Enhancer.
5. Add 10 µl Yeast Enzyme Salts and 80 µl Spheroplasting Enzyme Mixx. (Before first use, combine Spheroplasting Enzyme Solution with Spheroplasting Enzyme Mixx. Mix at room temperature for 5 minutes; aliquot into 80 µl fractions. The solution often appears 'grainy' at this point. Quick freeze in liquid nitrogen. Store at -70°C until ready to use; only thaw once).
6. Incubate at 37°C until spheroplast formation is complete (usually 12-30 minutes).

Start monitoring spheroplast formation after 12 minutes of incubation. Allowing the reaction to proceed after spheroplasting is complete is not beneficial to subsequent procedures.

Place 20 µl of Spheroplast Indicating Solution at one end of a glass slide and 20 µl of Spheroplast Control Solution at the other end. After the cells have incubated for 12 minutes, mix 2 µl of yeast cells with each solution; cover with cover slips, and observe under the microscope. Spheroplasting is sufficient when less than 5% of the cells remain intact in the visual field with the indicating solution, relative to the control solution.

At this point cells are easily lysed using the G NOME protocol.