LiAc-Mediated Yeast Transformation (long protocol)

In the LiAc transformation method, yeast competent cells are prepared and suspended in a LiAc solution with the plasmid DNA to be transformed, along with excess carrier DNA. Then a polyethylene glycol (PEG)/LiAc solution is added and the mixture is incubated at 30°C. After the incubation, DMSO is added and the cells are heat shocked, allowing the DNA to enter the cells. The cells are then plated on the appropriate medium to select for transformants containing the introduced plasmid(s). Because, in yeast, this selection is usually nutritional, an appropriate synthetic dropout (SD) medium is used.

1. Inoculate 5 mL of YPD or SD with a colony 2–3 mm in diameter from a fresh plate (no more then 3 weeks old) and grow overnight at 30˚C with shaking (16-18 hours).

2. Dilute culture to an OD600 of 0.2 (2x10e6 cells/mL) in 100 mL of fresh YPD or SD and continue to grow until the OD600 reaches 0.6. (NOTE: for many yeast strains a suspension containing 1 x 10e6 cells/mL will give an OD600 of 0.1).

3. Place cells in 50-ml tubes and centrifuge at 1000 x g for 5 minutes at RT.

4. Discard the supernatants and thoroughly resuspend the cell pellets in 25 mL of distilled`H2O. Pool and centrifuge again at 1000 x g for 5 minutes at RT.

5. Decant the supernatant and resuspend the cell pellet in 1 ml of freshly prepared, sterile 100mM LiAc in TE.

6. Transfer the cells to a 1.5 mL tube and centrifuge at top speed for 5 seconds. Remove the supernatant and add 500 uL of 100mM LiAc in TE (the cell titer should be ~3x10e9 cells/mL).

7. In a separate 1.5 mL tube add 0.1 ug of plasmid DNA and 100 ug of herring testes carrier DNA (NOTE: boil the carrier DNA for 5 minutes at 100˚C and chill immediately on ice before use. This insures that the DNA will be single stranded. It is not necessary to do it every time, rather every 3 freeze-thaw cycles for the aliquot in use. But always keep on ice when in use)

8. Add 100 uL of yeast competent cells to each tube and mix well by vortexing.

9. Add 600 uL of sterile freshly made PEG/LiAc (40% PEG 3350 + 100mM LiAc in 1x TE) solution to each tube and vortex at high speed for 10 seconds to mix.

10. Incubate at 30°C for 30 min with shaking at 200 rpm.

11. Add 70 ul of DMSO. Mix well by gentle inversion. Do not vortex.

12. Heat shock for 15 minutes in a 42°C water bath.

13. Chill cells on ice for 1–2 minutes.

14. Centrifuge cells for 5 seconds at 14,000 rpm at RT. Remove the supernatant and resuspend the cells in 0.5 ml of sterile 1xTE buffer.

[If using Geneticin® resuspend in 0.5ml media without Geneticin and incubate for at least 5 hours before plating on Geneticin containing plates.]

15. Plate 100 ul on the selection plates. To ensure plates with well-separated colonies, also spread 100 ul of a 1:1000, 1:100, and 1:10 dilution.

16. Incubate plates up side down at 30°C until colonies appear (generally, 2–4 days).

To calculate the transformation efficiency, count the colonies (cfu) growing on the dilution plate that has 30–300 cfu.

[cfu x resuspension vol. (uL)]/[Vol. plated (ul) x DF x DNA used (ug)]= cfu/ug DNA

The overall transformation efficiency should be at least 104 cfu/ug.

17. Pick the largest colonies and re-streak them on the same selection medium for master plates. Seal the plates with Parafilm and store at 4°C for 3–4 weeks.

The yeast transformation procedure described can be scaled up without a decrease in transformation efficiency.

Reagents:

10xTE buffer: 0.1 M Tris-HCl, 10 mM EDTA, pH 8.0. Autoclave.

50% PEG 3350 (Polyethylene glycol, avg. mol. wt. = 3,350; Sigma #P-3640): Prepare with sterile deionized H2O; if necessary, warm solution to 50°C to help the PEG go into solution. Filter sterile with a 0.45 um filter unit.

1M LiAc: 1 M lithium acetate adjusted to pH 7.5 with dilute acetic acid and autoclave.