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S.c. EasyComp™ Transformation Kit - FAQs

查看更多产品信息 S.c. EasyComp™ Transformation Kit - FAQs (K505001)

19 个常见问题解答

Pichia pastoris和S. cerevisiae的培养基有哪些不同种类？

以下是用于培养Pichia pastoris和S. cerevisia的营养丰富型和基本培养基：

营养丰富型培养基：

适用于S. cerevisiae和Pichia pastoris
•YPD（YEPD）：酵母提取物、蛋白胨和葡聚糖
•YPDS：酵母提取物、蛋白胨、葡聚糖和山梨醇

仅适用于Pichia pastoris
•BMGY：缓冲型甘油复合培养基
•BMMY：缓冲型甲醇复合培养基

基本培养基（又名缺陷型培养基）：

适用于S. cerevisiae
•SC（SD）：完全合成培养基（YNB、葡聚糖（或棉籽糖或半乳糖）以及氨基酸）

适用于Pichia pastoris

•MGY：基本甘油培养基
•MD：基本葡聚糖培养基
•MM：基本甲醇培养基
•BMGH：缓冲型基本甘油培养基
•BMMH：缓冲型基本甲醇培养基

Schizosaccharomyces pombe能否识别Saccharomyces cerevisiae的α-因子分泌信号？

当α因子基因中的P因子被替换为α时，S. pombe不能生成P因子。但是，当P因子基因中的α因子被替换为P时，S. pombe能够生成α因子。有反面证据表明，S. pombe能够加工其自身的交配因子切割位点，但并不是S. cerevisiae α因子的所有切割位点。最好使用一个更通用的信号序列（而不是前导信号序列，如α）。如果必须走前导路线，最好使用S. pombe的P因子前导而不是S. cerevisiae的α前导。

对于S. cerevisiae中的半乳糖诱导型表达，我能否在培养基中增加其他碳源以加速酵母生长，并且不抑制GAL启动子的表达？

在诱导期间，一些研究人员选择使酵母生长在以2%半乳糖作为唯一碳源的培养基中。但是，在含2%半乳糖和2%棉籽糖的诱导培养基中，酵母通常生长更快。棉籽糖是酵母的良好碳源，与葡萄糖不同，棉籽糖不会抑制GAL启动子的转录。棉籽糖是由半乳糖、葡萄糖和果糖依次连接而形成的三糖。大部分酵母可切割葡萄糖-果糖键，但不会切割半乳糖-葡萄糖键。果糖随后被用作碳源。

你们的试剂盒包含哪种S. cerevisiae酵母株？

我们提供INVSc1酵母株。这是一种二倍体酵母株，仅作表达用途。该酵母株不能形成良好的孢子，因此不适用于酵母遗传研究。INVSc1酵母株的基因型和表型如下：

基因型：MATa his3Δ1 leu2 trp1-289 ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-52
表型：His-, Leu-, Trp-, Ura-

注意：INVSc1缺乏组氨酸、亮氨酸、色氨酸和尿嘧啶。该酵母株在缺乏组氨酸、亮氨酸、色氨酸和尿嘧啶的SC基本培养基中不能生长。

旧的预混合醋酸锂缓冲液能否用于制备和转化Saccharomyces cerevisiae？

TE、醋酸锂和PEG的储液可保存。但是，制备待转化细胞的混合溶液，必须每次现用现配。如果溶液不是新配制的，则可能损失转化效率。

Saccharomyces cerevisiae培养物的光密度（OD）与细胞数的转换关系是什么？

OD600值为0.1，约等于3 x 106细胞/毫升。

在对Saccharomyces cerevisiae进行制备和电穿孔时，可达到的转化效率是多少？

转化效率很大程度上取决于酵母株，范围为1000-100,000个转化株/微克 DNA。

S. cerevisiae酵母转化有哪些不同方法？

以下是用于S. cerevisiae转化的不同方法：

•S. cerevisiae EasyComp转化试剂盒：易于使用的即用型试剂 ◦感受态细胞可冻存。转化效率高于103转化株/微克 DNA。与使用新鲜制备的细胞相比，使用冻存细胞可得到更高的转化效率。
•小规模酵母转化实验方案（见使用手册第13页）
•醋酸锂转化：简单、自己动手的实验方案 ◦感受态细胞必须是新鲜制备的
•电穿孔：简单、高效，自己动手的实验方案 ◦感受态细胞必须是新鲜制备的
•原生质球试剂盒：高效，工作量大，不适用于抗生素筛选

注意：选择适当的接种密度。菌落将在几天内出现。不要挑选最大的菌落，因为它们通常为抑制子。

What are the different kinds of media used for culturing Pichia pastoris and S. cerevisiae?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Will the Saccharomyces cerevisiae alpha-factor secretion signal be recognized by Schizosaccharomyces pombe?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

For galactose induction of expression in S. cerevisiae, can I include additional carbon sources in the media to increase yeast growth without repressing expression from the GAL promoter?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Which S. cerevisiae yeast strain do your kits contain?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can old premixed lithium acetate buffers be used for preparing and transforming Saccharomyces cerevisiae?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How does the optical density (OD) of a culture relate to the number of cells for Saccharomyces cerevisiae?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What range of efficiency of transformation I should expect when preparing and electroporating Saccharomyces cerevisiae?

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What are the different methods available for S. cerevisiae yeast transformation?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I sequenced one of your vectors after PCR amplification and observed a difference from what is provided online (or in the manual). Should I be concerned?

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.

Are your vectors routinely sequenced?

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

How was the reference sequence for your vectors created?

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