Sunday, November 05, 2006
hTOP2 beta Purification- Preparation of Yeast cells
1. Preparation of Yeast cells
- inoculate hTOP2beta/BCY123 into 50 ml of Ura- SD medium with 2% glucose and grow at 30oC for overnight.
- dilute cell culture 1:100 to Ura- SD medium with 3% glycerol and 1% lactic acid and grow for about another 12 hours.
- when OD 600 reach about 0.75, add final 2% galacotse to the culture (for induction of Galatose promoter)
- after induction by galactose for 6-14 hrs, harvest cells by 3000rpm spin for 5 min.
-wash cells once with ice-cold water and once with Buffer I.
- resuspend cells in Buffer I 1ml per 1g of cell pellete.
-freeze cells in liquide nitrogen and store frozen cells at -70oC.
## Buffer I:
50mM Tris pH 7.7/ 1mM EDTA/ 1mM EGTA/10 % glycerol/ 25 mM sodium fluoride/ 1mM sodium bisulfite/ 1mM PMSF/ 1mM b-mercaptoethanol/ 1mM benzamidine/ 5 ug/ml leupeptin/ 5ug/ml pepstatin.
- inoculate hTOP2beta/BCY123 into 50 ml of Ura- SD medium with 2% glucose and grow at 30oC for overnight.
- dilute cell culture 1:100 to Ura- SD medium with 3% glycerol and 1% lactic acid and grow for about another 12 hours.
- when OD 600 reach about 0.75, add final 2% galacotse to the culture (for induction of Galatose promoter)
- after induction by galactose for 6-14 hrs, harvest cells by 3000rpm spin for 5 min.
-wash cells once with ice-cold water and once with Buffer I.
- resuspend cells in Buffer I 1ml per 1g of cell pellete.
-freeze cells in liquide nitrogen and store frozen cells at -70oC.
## Buffer I:
50mM Tris pH 7.7/ 1mM EDTA/ 1mM EGTA/10 % glycerol/ 25 mM sodium fluoride/ 1mM sodium bisulfite/ 1mM PMSF/ 1mM b-mercaptoethanol/ 1mM benzamidine/ 5 ug/ml leupeptin/ 5ug/ml pepstatin.
# posted by yuanchin : 6:04 AM
