Transformation of Frozen Saccharomyces cerevisiae

Transformation of (Frozen) Competent Saccharomyces cerevisiae

1M Lithium Acetate: 10.2 g in 100 ml ddH2O – autoclave

50% PEG: Dissolve 50g in 30ml ddH2O and stir, bring total volume up to 100ml and heat and stir to combine. Filter sterilize (this might take about 10- 20 min). Bubbles will disappear once you filter sterilize.

ssDNA: Single-stranded salmon sperm carrier DNA is found in the –20C in its own box at a concentration of 1.5mg/ml in water (we do not sonicate). It must be boiled before use, but you can refreeze 3 times after the initial boil before you need to boil it again. Keep a few tubes in your own box for you personal use.

Requirements

1M Lithium Acetate 50% PEG ssDNA

Method

Spin down 100ul frozen competent cells from –80C(or made fresh) 1 minute at ~14,000 rpm. Use one tube per single or double transformation.

Aspirate supernatant and add the following in order.

  • 240ul 50% PEG
  • 36ul 1M LiAc
  • 79ul ssDNA
  • 5ul yourDNA

To give you a total solution of 360ul

A master mix may be made, by leaving out your DNA and mixing the other three ingredients, vortex lightly to mix before aliquotting. If doing a double transformation use 3ul of each of the DNA and reduce the amount of ssDNA to 78ul per transformation to keep the DNA/PEG-LiAc ratio the same.

Resuspend the yeast cells in the mixture by vortexing well to remove any clumps.

Incubate on rocker in 30oC room for 30 minutes.

Heat shock in 42oC water bath for 15 minutes.

Spin down at 14,000 rpm for 1 min., aspirate off the supernatant.

For double transformation resuspend cells in 100 ul ddH2O and plate out all cells. For single transformation resuspend cells in 200ul ddH2O and plate out half (100ul). Use appropriate media plates. (-trp, -trp -leu, etc…)

Incubate plates in 30oCvroom for 2-4 days until colonies appear.

This method is based, with permission, on an original protocol available here.

Tagged
comments powered by Disqus