McClean:Yeast Transformation

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Contents

Overview

Our lab's version of the Geitz lithium-acetate transformation method.

Materials

  • 1M LiAC
  • 100mM LiAC
  • 50% w/v PEG MW 3350 (Sigma P3640)
  • Sterile H20
  • Single-stranded carrier DNA (Sigma D1626, 2.0 mg/ml in TE buffer pH 8.0)
  • Appropriate selective plates (SC-URA, YPD+G418, etc)
  • Sterile 4mm glass-beads for plating (Fisher 11-312B)

Stock Solutions

Polyethylene glycol PEG 50% w/v (Sigma P3640)

  • Make up 50% w/v with H20 and filter-sterilize with a 0.45uM filter unit (Nalgene). We don't autoclave the PEG. Store in a tightly capped container to avoid evaporation.

Single-stranded carrier DNA (Sigma D1626)

  1. Weight out 200mg of the DNA into 100ml of TE buffer. Disperse the Dna into solution by drawing it p and dwn repeatedly in a 10-ml pipette. Mix vigorosly on a magnetic stirrer for 2-3 hours or until fully dissolved. Alternatively, leave the covered solution mixing at this stage overnight in a cold rom.
  2. Aliquot the DNA into 100μL portions and store at -20°C.
  3. Prior to use, the aliquot should be boiled and then quickly cooled on ice. We use a thermocycler to heat the DNA to 95°C for 25 minutes and then rapidly cool it on ice.

Once the salmon sperm has been boiled it can be freeze-thawed 3 or 4 times before transformation efficiencies begin to decrease. In practice, we boil the DNA before every transformation.

TE Buffer (pH 8.0)

  • 10 mM Tris-HCL (pH 8.0)
  • 1.0 mM EDTA

1.0M Lithium acetate stock solution (LiAc)

  • Prepare as a 1.0 M stock in distilled deionized H2O; filter-sterilize. The final pH should be between 8.4 and 8.9

Glass-beads for plating (Fisher 11-312B)

  • Pour beads into a small glass bottle (typically wide-mouthed 100ml or 250ml bottles work well) and autoclave on a 15 minute dry cycle to sterilize

Day 1

  1. Inoculate the strain to transform from a single colony into 5mls of YPD in a test tube. Put on the roller drum at 30°C overnight.

Day 2

  1. Inoculate 50 ml of YPD with 500 μL of the YPD overnight culture in a 250 ml flask. The 700 µl volume is approximate, and depends on the density of the strain you inoculate.
  2. Grow in shaking incubator for about 3-5 hours.
  3. Turn on 42°C water-bath (for heat-shock) if it is not already on.
  4. Harvest the cells by centrifuging in Eppendorf centrifuge model 5810R at 4000rpm (3130 xg) for 5 min. Resuspend pellet in 25 ml of sterile water by vortexing briefly. Pellet again and then resuspend in 1 ml of 100 mM LiAc.
  5. Transfer cell suspension to a 1.5 ml eppendorf tube, centrifuge at 3,000 xg for 2 min in an Eppendorf 5418 centrifuge and discard supernatant by removing it with a pipette.
  6. Add 400 µl 100 mM LiAc and resuspend cells by pipetting up and down. Aliquot 50 μL into 1.5 ml tubes (1 for each transformation). Pellet cells (3,000 xg for 2 min) and remove supernatant by aspiration.
  7. Add 300 μL T mix to each eppendorf tube of cells. Per one transformation reaction add '''IN ORDER''':
    • 240 μL 50% PEG 3350
    • 35 μL 1.0 M lithium acetate
    • 25 μL 2 mg/ml sssDNA
    • 50 μL sterile H20 and 20 μL of DNA (Note: You are aiming for a final concentration between 0.1-10 μg for plasmid DNA. Adjust your DNA and water amounts to add 70 μL of volume total)
  8. Vortex to resuspend cells.
  9. Incubate for 30 minutes at 30°C.
  10. Incubate tubes in a water bath at 42°C for 20-25 (up to 40) min. The time may need to optimized for your strain and transformation conditions.
  11. Microfuge at 3,000 xg for 15s, and remove transformation mix with a micropipette.
  12. Add 200 µL of sterile water to each tube and resuspend cells by pipetting it up and down as gently as possible if high efficiency is important.
  13. Plate your cells using glass beads:
    • Add 3-4 glass beads to each plate that you will be using.
    • Plate 150 µl of sterile water and add 20 µl cell suspension in one selection plate #1.
    • Plate the remaining 180 µl in selection plate #2
  14. Incubate at 30 °C. Colonies should appear after 2-4 days.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

Contact

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