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Spheroplasting 1. Harvest yeast at OD600 = 0.5 ( ~1-2 X 107 cells/ml).
 

Summary: 70
Spheroplasting
1. Harvest yeast at OD600 = 0.5 ( ~1-2 X 107 cells/ml).
2. Centrifuge (e.g., 5 minutes in IEC at 2000 rpm or in Sorvall GSA rotor at 5000 rpm).
3. Resuspend cells in dH2O. Centrifuge 5 minutes in IEC at 2000 rpm at 4C in tared tube.
4. Remove all of supernatant (decant and aspirate). Determine net weight yield of wet cell pellet.
5. Resuspend cell pellet in dH2O. Centrifuge again. Prepare pretreatment buffer.
6. Pretreatment: Resuspend per 1 g of wet weight in 4 ml of freshly prepared pretreatment buffer at
room temperature. Swirl intermittently for 10 minutes at room temperature.
100 ml Final
Pretreatment Buffer: 1 M Tris-HCl, pH 9 5 ml 50 mM Tris-HCl
DTT (BMB) 0.3 g 20 mM DTT
0.5 M EDTA, pH 8 1 ml 5 mM EDTA
7. Fill bottles with dH2O and centrifuge again at 4C.
8. Wash cell pellet with dH2O.
9. Wash cell pellet with 10 volumes of 5 mM KPi, pH 6.5.
10. Wash cell pellet with 10 volumes of 20% Sorbitol/5 mM KPi, pH 6.5.
11. Set up digestion as follows:
Per 1 gram
Digestion: 20% Sorbitol/KPi 4 ml

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine