Whole Cell Yeast PCR Kit Protocol
Revision No. 2069-999-7G01W

The kit is shipped at room temperature. However, upon receipt the Enzyme Mixx (lyophilized powder) should be stored at 4°C. After the Enzyme Mixx is mixed with the Enzyme Buffer the resulting WHOLE CELL LYSIS SOLUTION should be stored at 70°C in single use aliquots.

Prepare WHOLE CELL LYSIS SOLUTION and freeze in 100 µl single use aliquot at -70°C: Add Enzyme Buffer to Enzyme Mixx and dissolve at room temperature for no more than 10 minutes with intermittent agitation. Do not heat the solution which may appear grainy at this point. Aliquot (and flash freeze in liquid nitrogen, preferably) and store at 70°C. Thaw each aliquot only once and discard after use.

For yeast colonies or pelleted cells:

1. Touch a fresh yeast colony with a sterile disposable pipet tip and transfer to a microcentrifuge tube (or a 96 well microplate if processing multiple sample) containing 10 µg of WHOLE CELL LYSYS SOLUTION. Suspend the cells by gently pipetting up and down and/or gently swirling while avoiding the formation of bubbles.
   
   For yeast liquid colonies:
   
   Transfer 2 µl of log phase (2-5 x 10⁷/ml) cells to a tube (or microplate) containing 10 µl of WHOLE CELL LYSIS SOLUTION. Pipette up and down to ensure a homogeneous cell suspension.



2. Incubate at 37°C for 15 min. Mix by gently pipetting up and down with a wide bore pipet (or pipet tip with the end cut off) and use 2-5 µl for each 100 µl PCR reaction (include a 10 minute incubation step at 95°C before the PCR cycles). Store the remaining sample at -20°C.

No PCR signal

1. Inefficient cell lysis: increase the incubation time at 37°C for another 5 minutes and use a fresh yeast culture. You con check for cell wall removal by adding 2 µl of cells in WHOLE CELL LYSIS SOLUTION to 10 µl of Lysis Indicator and 10 µl to Control Solution and checking for cell lysis under the microscope. The majority of the cells (>80%) should be lysed when added to the Lysis Indicator.



2. Cells with damaged cell walls may have settled to the bottom of tube; resuspend before the PCR reaction.