MuLTEERatcliff Lab · Georgia Tech

Protocols / Transformation

Lithium acetate/PEG transformation of snowflake yeast

TransformationBy Various authorsVersion 1 · updated 2026-10-07

The standard LiOAc/PEG transformation adapted for multicellular clusters, for plasmids, PCR products or CRISPR constructs.

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Adapted from the lab compendium Snowflake Yeast Protocols — Ratcliff Lab / MuLTEE (V2).

Reagents (per sample)

Reagent Volume
PEG (50% w/w) 260 μL
LiOAc (1 M) 36 μL
ssDNA (2 mg/mL, freshly boiled) 50 μL
Digested plasmid/DNA 20 μL
ddH₂O 34 μL

Pre-culture

  1. Inoculate 2 mL from an overnight culture into 10 mL fresh growth medium.
  2. Grow the pre-culture for 3 hours.

Alternative: inoculate 1 mL from an overnight culture and grow for 4 hours instead of steps 1–2.

Cell prep

  1. Transfer 1 mL of pre-culture into a labeled 1.5 mL tube (one tube per line/population). Vortex briefly before use.
  2. Centrifuge at 8,000 rpm for 1 min.
  3. Confirm pellet volume reaches approximately the 100 μL mark (minimum halfway to 100 μL). If insufficient, add another 1 mL of culture to the same tube and re-spin.
  4. Remove supernatant without disturbing the pellet.
  5. Add 100 μL water and pipette up and down to break up clusters until the mixture is evenly suspended (light cream color, no visible individual colonies). Later-generation clusters require more vigorous pipetting.
  6. Add 900 μL water (1 mL total), centrifuge (8,000 rpm, 1 min), remove supernatant.
  7. Wash with 1 mL water a second time (centrifuge, discard supernatant).
  8. Wash with 1 mL 0.1 M LiOAc twice (dilute stock, not the 1 M stock used in Assembling the transformation mix). Each wash: add liquid, centrifuge, discard supernatant.
  9. Resuspend the final pellet in 100 μL 0.1 M LiOAc, pipetting to break up clusters.
  10. Centrifuge and remove supernatant thoroughly.

ssDNA prep (concurrent with LiOAc washes)

  1. Heat salmon sperm DNA (ssDNA) to 95 °C for 5 min.
  2. Set heat block to 42 °C immediately after.
  3. Mark the ssDNA tube each time it is boiled; do not reboil the same aliquot more than 3–4 times.
  4. Recover boiled ssDNA on ice quickly before using at room temperature
  5. Use ssDNA within ~30 min of boiling.

Assembling the transformation mix

Add to each cell pellet, in order:

  1. 260 μL PEG (50% w/w) — pipette slowly due to viscosity.
  2. 36 μL 1 M LiOAc — mix/swirl until uniformly cloudy.
    • Note specialized mixing technique:
      • Pipette up cells from bottom of solution and release at the top.
      • Swirl pipette around the sides of the tube on the way back down to the bottom to mix.
      • Repeat until large clusters crushed and solution uniform.
  3. 50 μL freshly boiled ssDNA (vortex briefly before adding).
  4. At least 20 μL DNA solution (plasmid, PCR product, etc.). For CRISPR: 10 μL Cas9 construct + 10 μL insert DNA. Mix before adding.
  5. ddH₂O as necessary so ddH2O + DNA solution add to 54 μL

Heat shock and recovery

  1. Incubate at 30 °C for 30 min with rotation.
  2. Heat-shock at 42 °C for 17 min (heat block, ~350 rpm if available).
  3. Centrifuge at 8,000 rpm for 2 min; remove all supernatant.
  4. Resuspend in 1.0 mL YPD, transfer to a fresh 2 mL tube, parafilm the lid closed to prevent lid popping open.
  5. Recover at 30 °C for 4 hours.
  6. Plate the desired volume (typically 100–500 μL) on selective medium.