Lithium Acetate Transformation
This protocol is originally from the
Cold Spring Harbor Laboratory Yeast Genetics & Genomics course manual.
Making Competent _S. cerevisiae Cells
This procedure makes enough competent cells for 5-10 transformations.
- Grow an overnight culture of each strain in YPD or selective media.
- The culture should be grown in flasks instead of tubes so the cells are properly aerated.
- Measure OD600:
- If OD600 is greater than or equal to 1.0, dilute cells back to 0.1 and grow for 4-6 hours.
- If OD600 is 0.2-1.0, use immediately or dilute for use later in the day.
- If OD600 is <0.2, continue growing cells.
- Transfer the cells to 50 ml conical tubes
- Pellet cells by centrifugation for 3-5 minutes at 2500 RPM.
- Remove media and resuspend the cell pellet in 10 ml of 1x LiOAc buffer by vortexing.
- Centrifuge the cells for 3 minutes at 2500 RPM.
- Remove liquid and resuspend pellet in 0.5-1.0 ml of 1x LiOAc buffer.
- Divide into 100 µl aliquots in Eppendorf tubes.
- Proceed to transformation.
Transforming S. cerevisiae Cells with Lithium Acetate
- Prepare single stranded carrier DNA.
- Thaw salmon sperm carrier DNA on ice, boil for 3 minutes, then cool on ice.
- Combine 100 µl of competent cells in 1x LiOAc buffer with: *10 µl of 10 mg/ml carrier DNA *1-5 µg of plasmid DNA or 25% of an efficient PCR (maximum 10 µl of total volume).
- Add 280 µl of PEG solution to each tube.
- Vortex or pipette vigorously to mix the PEG with other components.
- Incubate 20-45 minutes at room temperature.
- Add 43 µl of DMSO to each tube. Vortex or pipette to mix thoroughly.
- Incubate 5-15 minutes at 42°C.
- Immediately chill on ice for 3 minutes.
- Centrifuge cells for 2 minutes at half-speed (1250 RPM) and remove liquid.
- Add 0.5 ml of sterile water, vortex briefly, spin down, and remove liquid.
- For auxotrophic selection, resuspend in 200 µl of TE buffer. For antifungal, resuspend in 200 µl of YPD.
- Spread cells on appropriate selective plates and incubate for 2-3 days.
Notes
- This protocol yields ~500-5000 colonies/µg of plasmid DNA. Consider plating high and low volumes of cell suspension (e.g. 160 µl and 40 µl) to obtain plates with well-spaced transformants.
- You do not need to boil the carrier DNA every time. Keep a small aliquot in a -20°C freezer and re-boil after three or four freeze/thaw cycles.
- In our experience, incubating the cells with PEG for 45 minutes leads to a higher transformation efficiency.
- If cells are incubated at 42 °C for longer than 15 minutes, the cells may go into heat shock resulting in a failed transformation.
Materials
1x LiOAc buffer
- 0.1 M LiOAc
- 10 mM Tris-HCl (pH 8.0)
- X mM EDTA
1 M LiOAc
- Dissolve 51 g of LiOAc in 450 ml of water.
- Adjust volume to 500 ml.
- Filter sterilize or autoclave.
10x TE
- 10 ml 0.5 M EDTA
- 50 ml 1 M Tris HCl (pH 8.0)
- 440 ml sterile ddH2O
Single-stranded carrier DNA
- 10 mg/ml salmon sperm DNA
- 10 mM Tris-HCl (pH 8.0)
- 1 mM EDTA Boil for 3 minutes, cool on ice, and store at -20°C in aliquots.
PEG solution
- Heat 50 ml ddH2O in microwave.
- Add: * 50 g PEG 3350 * 10 ml 10x TE * 10 ml 1 M LiOAc
- Adjust the volume to 100 ml.
- Filter sterilize with a 0.45 µm filter unit.
- Store PEG solution in tightly capped container.
-- Main.DaciaLeon - 23 Feb 2017
Topic revision: r1 - 2017-02-23 - 21:16:11 - Main.DaciaLeon