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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