Yeast Transformation
1. Grow up 50 ml of cells overnight in YPD
2. Measure OD600…
- if OD600 is >1.0, dilute cells back to 0.1 and grow 4-6 hrs
- if OD600 is 0.2 – 1.0, cells can be used immediately or diluted for use
later in the day
- if OD600 is less than 0.2, continue growing cells
3. Spin down cells in 50 ml conical, 3 min. at setting 1/2 to 3/4
4. Meanwhile, remove salmon sperm DNA from freezer and place in 100 degree
sand bath
5. Pour off media and resuspend pellet in 5 ml sterile TE by vortexing
6. Spin down cells, 3 min.
7. Pour off TE and resuspend pellet in 5 ml LiOAc mix by vortexing
8. Spin down cells, 3 min.
9. Pour off LiOAc mix and resuspend pellet in 0.5 – 1 ml LiOAc mix
10. In a 1.5 ml eppie mix:
1-5 g DNA in 10-50 l (remember to do a no DNA control too)*
10 l salmon sperm DNA, freshly boiled
100 l cells in LiOAc mix
11. Add 700 l PEG mix to each
12. Vortex briefly to resuspend cells
13. Incubate 30 min. at room temperature
14. Add 48 l DMSO to each
15. Vortex briefly
16. Incubate 15 min. at 42 degrees
17. Spin down 1 min. 5K in microfuge
18. Aspirate off liquid
19. Add 200-500 l YPD
20. Spread onto appropriate selective plates**
21. Incubate 2 days at 30 degrees (non-ts strains) or 3 days at 23 degrees (ts strains)
* 2-micron and Cen-based plasmids can be directly transformed; integrating plasmids
must be cut with a restriction enzyme to target them for integration into the yeast
genome; PCR products can be directly transformed
Good things to cut with to target integration at marker loci. But check that the enzyme
does not cut in your insert!
URA3: StuI, NcoI, NdeI (EcoRV if it’s been deleted from poly-linker)
LEU2: XcmI, AflII, BstEII @ 60°, AgeI
TRP1: Bsu36I, BstZ17I, SnaBI, MfeI
HIS3: NheI, MscI, NdeI
ADE2: StuI, HpaI, AflII, AatII
LYS2: StuI, SexAI, BstZ17I, HpaI
** plate directly to amino-acid drop out plates; to select for KANMX, plate to YPD
overnight then replica plate to YPD + geneticin the next day