S.c. EasyComp™ Transformation Kit - FAQs

Following are the rich and minimal media used for culturing Pichia pastoris and S. cerevisiae:

Rich Media:
S. cerevisiae and Pichia pastoris
YPD (YEPD): yeast extract, peptone, and dextrose
YPDS: yeast extract, peptone, dextrose, and sorbitol

Pichia pastoris only
BMGY: buffered glycerol-complex medium
BMMY: buffered methanol-complex medium

Minimal Media (also known as drop-out media):
S. cerevisiae
SC (SD): Synthetic complete (YNB, dextrose (or raffinose or galactose), and amino acids)

Pichia pastoris
MGY: minimal glycerol medium
MD: minimal dextrose
MM: minimal methanol
BMGH: buffered minimal glycerol
BMMH: buffered minimal methanol

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S. pombe cannot generate P factor when P factor is replaced for alpha in the alpha factor gene. It can, however, produce alpha factor when alpha is replaced for P in the P factor gene. This is negative evidence that S. pombe can process its own mating factor cleavage sites, but not all the cleavage sites of the S. cerevisiae alpha factor. It is better to use a more generic signal sequence (rather than a pre- pro- signal sequence such as alpha). If it is necessary to go the pre- pro- route, it is better to use the S. pombe P factor leader rather than the S. cerevisiae alpha leader.

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Some researchers choose to grow yeast in medium containing 2% galactose as the sole carbon source during induction. However, yeast typically grow more quickly in induction medium containing 2% galactose plus 2% raffinose. Raffinose is a good carbon source for yeast, and unlike glucose, does not repress transcription from the GAL promoter. Raffinose is a trisaccharide of galactose, glucose, and fructose linked in that order. Most yeast can cleave the glucose-fructose bond, but not the galactose-glucose bond. Fructose is then used as a carbon source.

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We offer the INVSc1 yeast strain. It is a diploid strain for expression purposes only. It does not sporulate well and is therefore not suited for yeast genetic studies. The genotype and phenotype of the INVSc1 strain are as follows:

Genotype: MATa his3D1 leu2 trp1-289 ura3-52/MATalpha his3D1 leu2 trp1-289 ura3-52
Phenotype: His-, Leu-, Trp-, Ura-
Note that INVSc1 is auxotrophic for histidine, leucine, tryptophan, and uracil. The strain will not grow in SC minimal medium that is deficient in histidine, leucine, tryptophan, and uracil.

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Stock buffers of TE, lithium acetate, and PEG can be stored. However, the combined solution used to prepare the cells for transformation must be made fresh every time. There is a loss in transformation efficiency if the solutions are not freshly prepared.

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OD600 of 0.1 = approximately 3 x 10e6 cells/mL

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The efficiency is very strain-dependent, but 1000 to 100,000 transformants per µg DNA is the range.

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Here are the different methods available for S. cerevisiae transformation:

- S. cerevisiae EasyComp Transformation Kit (K505001): easy-to-use, ready-made reagents
Competent cells can be stored frozen. Transformation efficiency is >10e3 transformants per µg DNA. Higher transformation efficiencies are often obtained with frozen versus freshly prepared cells.
- Small-scale yeast transformation protocol (page 13 of the manual)
- Lithium acetate transformation: easy, do-it-yourself protocol
Competent cells must be made fresh
- Electroporation: easy and high efficiency, do-it-yourself protocol
Competent cells must be made fresh
- Spheroplast Kit for Yeast (K172001): high efficiency, a lot of work, not suitable for antibiotic selection
Note: Plate an appropriate density. Colonies will appear over several days. Don't pick the largest colonies, as these are often suppressors.

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Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.

No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.

Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.