Lithium acetate Dihydrate: Yeast Transformation Protocol

Lithium acetate dihydrate is a widely used chemical reagent that enables efficient introduction of exogenous DNA into Saccharomyces cerevisiae by transiently permeabilizing the yeast cell wall and membrane.

Chemical Name

Lithium acetate dihydrate

Molecular Formula

C₂H₃LiO₂·2H₂O

Molecular Weight

102.02 g/mol

CAS Number

6108-17-4

SKU

L755673

Primary Application

Yeast (S. cerevisiae) transformation via the lithium acetate method

Working Concentration

100 mM LiOAc (typical transformation buffer)

Background and Principle

The lithium acetate (LiOAc) method is the most widely adopted technique for introducing plasmid DNA or linear DNA cassettes into Saccharomyces cerevisiae. Lithium acetate dihydrate dissociates in aqueous solution to provide lithium cations (Li⁺) and acetate anions. The monovalent Li⁺ ion is thought to interact with the cell wall and plasma membrane of yeast, temporarily increasing permeability and allowing exogenous DNA to enter the cell.

The transformation efficiency of the LiOAc method is substantially enhanced by the co-addition of single-stranded carrier DNA (typically heat-denatured salmon sperm or herring sperm DNA) and polyethylene glycol (PEG). Carrier DNA is believed to compete with transforming DNA for non-specific binding sites on the cell surface, while PEG promotes cell-DNA contact and protects DNA from nuclease degradation. A brief heat-shock step (typically at 42 °C) further increases uptake efficiency.

Lithium acetate dihydrate (Aladdin, SKU: L755673) provides a convenient, pre-weighed anhydrous-equivalent source of lithium acetate with well-defined stoichiometry, making buffer preparation reproducible and straightforward.

Materials and Reagent Preparation

Materials Required

• Lithium acetate dihydrate (Aladdin L755673)
• Polyethylene glycol (PEG 3350 or PEG 4000), 50% (w/v) solution
• Single-stranded carrier DNA (e.g., salmon sperm DNA, 2 mg/mL, heat-denatured)
• Plasmid or linear DNA of interest (≥1 µg per transformation)
• Sterile deionized water (dH₂O)
• YPD or appropriate selective liquid medium
• Selective agar plates
• 1.5 mL microcentrifuge tubes
• Water bath or heat block set to 30 °C and 42 °C
• Microcentrifuge

Solution Preparation

1 M Lithium Acetate Stock Solution: Dissolve 10.20 g of lithium acetate dihydrate (L755673) in 80 mL of sterile dH₂O. Adjust volume to 100 mL. Sterilize by autoclaving or filter-sterilization (0.22 µm). Store at room temperature.

100 mM LiOAc Working Solution: Dilute 1 M LiOAc stock 1:10 in sterile dH₂O.

Carrier DNA: Prepare salmon sperm DNA at 2 mg/mL in TE buffer. Boil for 5 minutes, then immediately cool on ice before each use. Store in aliquots at −20 °C.

PEG/LiOAc Mix (transformation mix): Combine 240 µL of 50% PEG (w/v), 36 µL of 1 M LiOAc, and sterile dH₂O as required per transformation reaction.

Step-by-Step Transformation Protocol

Day 1 — Culture Preparation

1. Inoculate a single colony of S. cerevisiae into 5 mL YPD and grow overnight at 30 °C with shaking (200 rpm).

Day 2 — Transformation

1. Sub-culture: Dilute overnight culture into fresh YPD to an OD₆₀₀ of approximately 0.2. Incubate at 30 °C with shaking until OD₆₀₀ reaches 0.6–0.8 (approximately 3–4 hours). This ensures cells are in mid-log phase.
2. Harvest cells: Transfer the appropriate volume of culture to a microcentrifuge tube. Pellet cells by centrifugation at 3,000 × g for 5 minutes. Discard supernatant.
3. Wash with LiOAc: Resuspend the cell pellet in 1 mL of 100 mM LiOAc (prepared from L755673). Centrifuge at 3,000 × g for 5 minutes. Discard supernatant.
4. Prepare transformation mix: For each transformation, add in the following order to the cell pellet: 240 µL 50% PEG, 36 µL 1 M LiOAc, 25 µL boiled carrier DNA (2 mg/mL), and DNA of interest (1–5 µg) in up to 50 µL dH₂O.
5. Vortex: Mix vigorously by vortexing for 1 minute until cell pellet is fully resuspended.
6. Incubate at 30 °C: Incubate the reaction at 30 °C for 30 minutes.
7. Heat shock: Transfer tubes to a 42 °C water bath or heat block for 25–40 minutes. Do not exceed 42 °C.
8. Recovery: Centrifuge at 3,000 × g for 1 minute. Remove and discard the supernatant.
9. Resuspend and plate: Resuspend the pellet in 100–200 µL of sterile dH₂O or YPD. Spread onto selective agar plates.
10. Incubate plates: Incubate at 30 °C for 2–4 days until colonies appear.

Troubleshooting and Optimization Tips

Low or no transformants:

• Ensure cells are harvested at mid-log phase (OD₆₀₀ 0.6–0.8). Stationary-phase cells transform poorly.
• Verify that carrier DNA has been freshly boiled and immediately chilled on ice.
• Check that the PEG solution has not degraded; old or impure PEG significantly reduces efficiency.
• Confirm that the lithium acetate dihydrate (L755673) solution is at the correct concentration (100 mM working, 1 M stock).
• Increase DNA quantity (up to 5 µg) or use higher-purity DNA preparation.

Contamination on plates:

• Use freshly prepared selective plates and ensure all reagents are sterilized.

Poor reproducibility:

• Standardize the heat-shock duration and temperature carefully. Variation in 42 °C heat-shock time is a common source of variability.
• Use freshly prepared 1 M LiOAc stock from lithium acetate dihydrate (L755673) for each experiment where possible.

Storage of reagents: Lithium acetate dihydrate is hygroscopic. Store the solid reagent tightly sealed at room temperature and weigh quickly to minimize moisture uptake.

Safety, Handling, and Disposal

Lithium acetate dihydrate is considered a low-hazard chemical under standard laboratory conditions; however, appropriate precautions should always be observed:

• Personal Protective Equipment (PPE): Wear laboratory coat, chemical-splash goggles, and nitrile gloves when handling the solid or concentrated solutions.
• Skin and Eye Contact: In case of skin or eye contact, flush immediately with large amounts of water for at least 15 minutes and seek medical advice if irritation persists.
• Ingestion/Inhalation: Avoid generating dust when weighing. Do not ingest. Lithium salts may pose systemic toxicity if ingested in significant quantities.
• Disposal: Dilute aqueous solutions may generally be disposed of via the drain in accordance with local regulations. Consult your institution's Environmental Health and Safety (EHS) office for guidance on lithium-containing waste disposal.
• Storage: Store tightly closed at room temperature (15–25 °C), away from moisture and incompatible materials (strong oxidizers, strong acids).

Always consult the Safety Data Sheet (SDS) for Aladdin product L755673 before use.

Frequently asked questions

Why is lithium acetate dihydrate used instead of anhydrous lithium acetate for yeast transformation?

Lithium acetate dihydrate (MW 102.02 g/mol) is a stable, freely soluble form that is easier to weigh accurately and store compared to anhydrous lithium acetate. Because the water of crystallization is fixed and defined, stock solutions can be prepared with high concentration accuracy. Either form can be used; the dihydrate simply requires a correction in the amount weighed to achieve the same molar concentration.

What is the recommended concentration of lithium acetate for the yeast transformation buffer?

The standard working concentration is 100 mM lithium acetate. This is prepared by diluting a 1 M lithium acetate stock solution (made from lithium acetate dihydrate, Aladdin L755673) ten-fold in sterile deionized water. Higher concentrations may be toxic to yeast cells, while lower concentrations reduce transformation efficiency.

Can lithium acetate dihydrate be used for transformation of organisms other than Saccharomyces cerevisiae?

Yes. The lithium acetate method has been adapted for transformation of other yeasts, including Pichia pastoris (Komagataella phaffii) and Schizosaccharomyces pombe, as well as some filamentous fungi. However, the protocol conditions (LiOAc concentration, heat-shock temperature and duration) may need to be optimized for each organism.

How should lithium acetate dihydrate be stored to maintain its quality?

Lithium acetate dihydrate is hygroscopic and should be stored in a tightly sealed container at room temperature (15–25 °C), away from moisture, heat, and incompatible materials such as strong acids and oxidizers. Always reseal the container promptly after use to prevent caking or changes in stoichiometry due to moisture absorption.

How much lithium acetate dihydrate is needed to prepare 100 mL of 1 M stock solution?

To prepare 100 mL of 1 M lithium acetate solution using the dihydrate form (MW = 102.02 g/mol), dissolve 10.20 g of lithium acetate dihydrate (Aladdin L755673) in approximately 80 mL of sterile deionized water, then adjust the volume to 100 mL. Sterilize by autoclaving or filtration through a 0.22 µm membrane.

References

1. Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols. 2007;2(1):31–34. doi:10.1038/nprot.2007.13
2. Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. Journal of Bacteriology. 1983;153(1):163–168. PMID:6336730
3. Gietz RD, Woods RA. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods in Enzymology. 2002;350:87–96. PMID:12073338

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