All steps centrifugation steps are performed in a microfuge unless otherwise noted.

 

  1. Grow 25mL yeast in YES or selective media to OD595=0.5-1.

 

  1. Harvest the cells by centrifugation in a clinical centrifuge (Allegra, 5min, 2krpm)

 

  1. Resuspend cells in STOP-Buffer.

 

  1. Wash the cells in 1mL of Buffer S.

 

  1. Re-suspend the cells in 0.60mL of Buffer S + 1mM PMSF + 10mM b-mercaptoethanol.

 

  1. Incubate cells at 30C for 10 min.

 

  1. Pellet cells by centrifugation.

 

  1. Re-suspend cells in 4X pellet volume of buffer S + 1mM PMSF + Zymolyase (~50-100ug/mL). (I have found that the greater the amounts of zymolyase the better (ie ~1mg zymo/1mg cell pellet).

 

  1. Incubate at 30C for 40 min. Cells can be viewed under the microscope to monitor cell wall degradation, however I have found it easier to simply incubate for 40 min and than continue.

 

  ***Remaining steps must be performed on ice, buffers must be pre-chilled.

 

  1. Dilute cells with 0.7mL of buffer S + 1mM PMSF.

 

  1. Pellet cells by gentle centrifugation. (~3-5Krpm for 2min).

 

  1. Wash cells 1X in Buffer S + 1mM PMSF

 

  1. Re-suspend final cell pellet in 0.3mL Buffer F.

 

  1. Lyse cells by homogenization. I use a teflon pestel in a microfuge tube but a dounce homogenizer can also be used. Monitor lysis under microscope (number of steps of homogenization can vary, I use 7-10 strokes 2-3 times with 0.5-1 min. icing in between).

 

  1. Pellet un-lysed cells by gentle centrifugation. (3 krpm for 5 min. although I use 3 krpm in 1 min increments, monitoring removal of un-lysed cells by microscopy between steps: I have found that the removal of un-lysed cells can to vary from strain to strain. It is important to not spin too high so that nuclei do not spin out in the pellet).

 

  1. Using a tip with the end cut off, gently pipet the lysate onto 0.2mL of buffer GF in a microfuge tube. Centrifuge for 2 min at 6-8 Krpm.

 

  1. Remove 100-200 uL of cytoplasmic lysate (supernatant). Aspirate off remaining supernatant with a vacuum syringe. (Removal of the cytoplasmic lysate in this manner prevents disruption of the nuclear pellet and prevents cytoplasmic 'spillover'.)

 

  1. Resuspend nuclei pellet in a suitable volume of lysis buffer.

 

  Buffer S:

  1.4M Sorbitol

  40mM HEPES (pH=7.2)

  0.5mM MgCl2

  (sterilize by filtration)

 

  Buffer F

  18% Ficoll 400, w/v

  20mM HEPES (pH=7.2)

  0.5mM MgCl2

  Protease inhibitors (PMSF, pepstatin, leupeptin, aprotinin, benzamidine)

 

  Buffer GF

  7% Ficoll 400, w/v

  20% glycerol

  20mM HEPES (pH=7.2)

  0.5mM MgCl2

  Protease inhibitors