Yeast Transformation Protocol
Yeast Transformation Protocol
1. Grow up preculture 1-2x ON in media.
2. Take OD660 preculture.
3. Dilute cells to an OD660 of ~0.390 in 50-100 ml media (YPD).
4. Grow for ~3 - 5 hrs. until cells have an OD660 of ~1.0.
5. Spin down cells 3000 x G for 5min and decant supernatant (keep pellet).
6. Resuspend with 25ml TE/LiOAC.
7. Spin down 3000xG for 5min and decant supernatant (keep pellet).
8. Resuspend pellet in 1 ml TE/LiOAC – cells may be kept for ~ a week at 4(C.
9. To each eppendorf tube add 50 (l cells + 5 (l transforming DNA + 5(l carrier DNA
(Salmon Sperm DNA – 0.1 mg/ml-heat for 5min at 100C).
10. Add 500 (l of PEG mix to each tube and vortex for 10 seconds.
11. Incubate at 30(C for 30 minutes.
12. Heat shock at 42(C for 15 minutes.
13. Spin down cells 6000rpm/1min and resuspend in amino acid solution (100ul ddH20 is fine).
Ade – 250 (l/tube His – 75 (l/tube Leu – 75 (l/tube
Lys – 75 (l/tube Trp – 75 (l/tube Ura – 250 (l/tube
13. Plate cells and incubate at 30(C.
10x TE: 12.10 g Tris (
3.68 g EDTA
1000 ml dH2O
1000 ml
10x LiOAC – 1M LiOAC pH 7.5: 20.40 g LiOAC
200 ml dH2O ( Autoclave
200 ml
TE/LiOAC: 20.0 ml 10x TE
20.0 ml 10x LiOAC
160 ml dH2O
200 ml (
PEG 50% (w/vol) 250 g PEG (MW 3350) Filter Sterile
500 ml dH2O
500 ml
Final Concentration
PEG mix 200 ml 50% PEG 40%
25 ml 10x TE 1x
25 ml 10x LiOAC 1x
250 ml
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