Home
Resources
Publications
High Efficiency Transformation of Schizosaccharomyces Pombe Pretreated With Thiol Compounds by Electroporation

High Efficiency Transformation of Schizosaccharomyces Pombe Pretreated With Thiol Compounds by Electroporation

M Suga, T Hatakeyama

Yeast. 2001 Aug;18(11):1015-21.

PMID: 11481672

Abstract:

A highly efficient method for transformation of the fission yeast Schizosaccharomyces pombe by electroporation has been developed. Significantly higher transformation efficiency was obtained when intact cells grown in SD medium (0.67% Bacto yeast nitrogen base without amino acids, 2% glucose) were pretreated with thiol compounds before an electric pulse was applied to the cells. Among the thiol compounds tested, dithiothreitol (DTT) was the most effective for pretreatment. A high transformation efficiency was obtained when the cells were pretreated with 25 mM DTT at 30 degrees C for 15 min in an osmotically adjusted buffer, since the cells were sensitive to osmotic pressure. It was important to exclude glucose from the DTT pretreatment buffer, as it caused a drastic decrease in efficiency. The optimal cell concentration and amount of DNA during the electric pulse were 1x10(9) cells/ml and 10 ng, respectively. The maximum transformation efficiency, 1.2x10(7) transformants/microg plasmid DNA, was obtained when an electric pulse of 11.0 kV/cm was applied for 5 ms. Furthermore, the high competency of cells pretreated with DTT was maintained by freezing them in a non-permeating cryoprotectant such as sorbitol.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411447	Yeast Nitrogen Base Without Amino Acids			Price

As a specialist in analytical chemical Products, Alfa Chemistry offers consumers a wealth of raw materials and a full range of technologies and services.

QUICK LINKS

HEADQUARTERS

Tel:

Fax:

Email: