Yeast transformation Cell Competency and Transformation Protocol
Yeast transformation Cell Competency and Transformation Protocol
(Schiestl & Gietz)
1. Pipette 5 ml of dropout media into a sterile 17 x 100 mm, 14 ml polypropylene, round bottom tube (e.g. Falcon). Inoculate media with yeast from a growing colony. Shake in 30°C incubator at 250 rpm overnight. You may want to inoculate several tubes -- use the one which grows fastest for the remainder of the procedure.
2. Vortex the yeast tube (to disaggregate HF7c yeast strains) and add the contents to a sterile 125 ml flask containing 45 ml of YPD. Shake the flask containing the 50 ml mixture in a 30°C incubator at 250 rpm for 5-12 hrs (optimal incubation time is empirically-derived).
3. Pour the contents of the flask into a sterile 50 ml Corning centrifuge tube and place the tube into a fixed-angle rotor within centrifuge. Balance tube and spin at 7000 rpm for 5 min. Pour off supernatant.
4. Resuspend pellet in 50 ml of ddH2O by vortexing. Repeat centrifugation at 7000 rpm for 5 min.
5. To aspirate all of supernatant without disturbing pellet: discard supernatant by inverting 50 ml tube and keeping it inverted, pick up a micropipetter with the volume set to 1000 l. Immediately after positioning the 50 ml tube upright, suck out any residual liquid that gravitates to the tube bottom (usually around a few hundred l).
6. Resuspend pellet in 1 ml of 100mM LiAc and transfer resuspension to a microfuge tube.
7. Spin microfuge tube on high for 20 sec and aspirate supernatant.
8. Add a volume of 100mM LiAc to pellet to bring solution to a final volume of 0.5 ml (usually around 400 l of 100mM LiAc).
9. Thoroughly resuspend pellet by vortexing and/or micropipetting up and down.
10. Prepare a 50 l aliquot in a microfuge tube for each transformation desired.
11. Spin microfuge tube on high for 20 sec and carefully aspirate supernatant.
12. Add transformation indgredients to pelleted yeast in this order:
1. 240 l of 50% PEG (buffers cells from 1M LiAc)
2. 36 l of 1M LiAc
3. 25 l of Salmon Sperm DNA (I incubate this at 95°C for five minutes before using)
4. 50 l of plasmid/ddH2O solution (I use 50 l of plasmid when library screening)
13. Vortex tube ~30 sec to resuspend pellet in transformation mixture.
14. Incubate tube 30 min at 30°C.
15. Heat shock tube 20-25 min at 42°C.
16. Spin microfuge tube at 8000 rpm and carefully aspirate supernatant (don't want to lose any transformed yeast).
17. Very gently resuspend pellet in 1 ml of ddH2O by micropipetting up and down.
18. Plate on dropout media. Spread just enough to expedite uptake of liquid by the media. If plating on 150 mm plates, spread 250 l of transformed yeast suspension.
If plating on 100 mm plates, spread 100 l of suspension along with 100 l of ddH2O.
19. Incubate plates at 30°C for 3-5 days and look for colonies.
(Schiestl & Gietz)
1. Pipette 5 ml of dropout media into a sterile 17 x 100 mm, 14 ml polypropylene, round bottom tube (e.g. Falcon). Inoculate media with yeast from a growing colony. Shake in 30°C incubator at 250 rpm overnight. You may want to inoculate several tubes -- use the one which grows fastest for the remainder of the procedure.
2. Vortex the yeast tube (to disaggregate HF7c yeast strains) and add the contents to a sterile 125 ml flask containing 45 ml of YPD. Shake the flask containing the 50 ml mixture in a 30°C incubator at 250 rpm for 5-12 hrs (optimal incubation time is empirically-derived).
3. Pour the contents of the flask into a sterile 50 ml Corning centrifuge tube and place the tube into a fixed-angle rotor within centrifuge. Balance tube and spin at 7000 rpm for 5 min. Pour off supernatant.
4. Resuspend pellet in 50 ml of ddH2O by vortexing. Repeat centrifugation at 7000 rpm for 5 min.
5. To aspirate all of supernatant without disturbing pellet: discard supernatant by inverting 50 ml tube and keeping it inverted, pick up a micropipetter with the volume set to 1000 l. Immediately after positioning the 50 ml tube upright, suck out any residual liquid that gravitates to the tube bottom (usually around a few hundred l).
6. Resuspend pellet in 1 ml of 100mM LiAc and transfer resuspension to a microfuge tube.
7. Spin microfuge tube on high for 20 sec and aspirate supernatant.
8. Add a volume of 100mM LiAc to pellet to bring solution to a final volume of 0.5 ml (usually around 400 l of 100mM LiAc).
9. Thoroughly resuspend pellet by vortexing and/or micropipetting up and down.
10. Prepare a 50 l aliquot in a microfuge tube for each transformation desired.
11. Spin microfuge tube on high for 20 sec and carefully aspirate supernatant.
12. Add transformation indgredients to pelleted yeast in this order:
1. 240 l of 50% PEG (buffers cells from 1M LiAc)
2. 36 l of 1M LiAc
3. 25 l of Salmon Sperm DNA (I incubate this at 95°C for five minutes before using)
4. 50 l of plasmid/ddH2O solution (I use 50 l of plasmid when library screening)
13. Vortex tube ~30 sec to resuspend pellet in transformation mixture.
14. Incubate tube 30 min at 30°C.
15. Heat shock tube 20-25 min at 42°C.
16. Spin microfuge tube at 8000 rpm and carefully aspirate supernatant (don't want to lose any transformed yeast).
17. Very gently resuspend pellet in 1 ml of ddH2O by micropipetting up and down.
18. Plate on dropout media. Spread just enough to expedite uptake of liquid by the media. If plating on 150 mm plates, spread 250 l of transformed yeast suspension.
If plating on 100 mm plates, spread 100 l of suspension along with 100 l of ddH2O.
19. Incubate plates at 30°C for 3-5 days and look for colonies.
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