The Frozen-EZ Yeast Transformation II Kit is designed to make yeast transformations and library screening easier and more efficient than currently available methods. The yeast cells prepared with the Frozen-EZ Yeast Transformation II Kit can be used immediately for transformation or can be stored (i.e., = -70°C) for use at a later time. Yeast prepared with this kit can be transformed with both circular and linear DNAs. Also, the Frozen-EZ Yeast Transformation II Kit can be used with other fungi including C. albicans, S. pombe, and P. pastors.
Q1: What is in this kit and how does it work?
The procedure utilized in this kit is designed, in some ways, similar to the lithium cation based method. No spheroplast step is involved. The mechanism probably involves some metabolic pathways that we do not fully understand.
Q2: Does this kit work on C. albicans, S. pombe, or Pichia pastoris?
Yes. Based on data from other labs and ours, this kit does work as well on C. albicans, S. pombe, or Pichia pastoris as well as S. cerevisiae.
Q3: How long can I store my competent yeast cells below -70°C?
There is no loss of transformation efficiency after half a year of storage below -70°C. The transformation efficiency gradually starts to drop after 6 months.
Q4: How should I thaw the stored frozen competent cells?
Thaw at room temperature.
Q5: Do the frequencies of freezing and thawing affect the transformation efficiency?
We usually see a 10-30% increase of transformants after the first cycle of freezing. You can refreeze and thaw the competent yeast cells 3-4 times without noticeable effect on the transformation efficiency. Further freezing-thawing cycles adversely affect the transformation efficiency.
Q6: In step 2 of the transformation, do I need to incubate strictly at 30°C?
No. Temperatures between 30°C and 37°C are in the optimal temperature range. Incubation below or above this range greatly reduces the transformation efficiency.
Q7: Can I use DNA directly from restriction enzyme digestions without purification?
Yes. Different digestion buffers have only a slight effect on the transformation efficiency. You should try to keep the DNA volume in 5 µl per transformation experiment by increasing the concentration of DNA in the digestion reaction.
Q8: How much transformation mixture should I spread on a selective plate?
For most circular plasmid transformations, 50 µl is enough. But if you use linearized DNA or use more than one selection marker, you can apply up to 200 µl of transformation mixture on each plate to increase the number of transformants. The number of transformants increases linearly with the amount of transformation mixture applied to each plate.
Q9: Do I need to use plates with sorbitol?
No. Use any plate that is appropriate for your experiment.
Q10: Do I need to add carrier DNA?
No. There is no need for carrier DNA.
