Yeast transformation
LiSorb(filter sterilize)
100 mM LiOAc
10 mM Tris-HCl (pH 8.0)
1 mM EDTA
1 M Sorbitol
LiPEG (filter sterilize)
100 mM LiOAc
10 mM Tris-HCl (pH 8.0)
1 mM EDTA
40% PEG3350
手順
Grow cells over night in 50 ml of appropriate medium to an OD600 of 0.3 to 1.5.
↓ 3200 rpm, 5 min
Remove supernatant. Wash with 50 ml d.w.
↓ 3200 rpm, 5 min
Remove supernatant. Wash with 10 ml LiSorb.
↓ 3200 rpm, 5 min
Remove supernatant and resuspend cells in 300 ul of LiSorb.
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Aliquot cells in 50 ul portions. Add 5 ul of denaturated 10 mg/ml carrier DNA (heat 5 min at 95゚C to denature DNA, place on ice for a couple of seconds).
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Mix cells and incubate for 20 min at RT.
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Add 35 ul DMSO. Vortex. Heat shock cells for 5-15 min at 42゚C. (Wild type: 15 min, ts-mutants: 5 min.)
↓ 1500 rpm, 3 min, 4゚C
Remove supernatant. Add 300 ul appropriate medium or PBS (steril).
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Resuspend and plate out on selective plate.
(For G418-selection: the cells were resuspended in 3 ml of liquid YPDA, shaked for 3 hrs at 30゚C, before spreading them on slective G418 plates. Hygromycin for 8 hrs.)