McClean:Yeast Transformation

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Revision as of 13:46, 20 July 2011 by Megan N McClean (Talk | contribs)
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Contents

Overview

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

Materials

  1. 1M LiAC
  2. 100mM LiAC
  3. 50% w/v PEG MW 3350 (Sigma P3640)
  4. Sterile H20
  5. Single-stranded carrier DNA (Sigma D1626, 2.0 mg/ml in TE buffer pH 8.0)
  6. Appropriate selective plates (SC-URA, YPD+G418, etc)

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 acetat 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

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

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

  • Who has experience with this protocol?

or instead, discuss this protocol.


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