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  • Highly-efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants.

Highly-efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants.

Yeast (Chichester, England) (2012-10-16)
Xinping Lin, Fan Yang, Yongjin Zhou, Zhiwei Zhu, Guojie Jin, Sufang Zhang, Zongbao Kent Zhao
ABSTRACT

Red yeasts hold great promise in the production of microbial lipids and carotenoids. Genetic study of red yeasts has attracted much attention; however, rapid amplification of genes from red yeast samples remains technically challenging. Here a highly efficient method for the preparation of genomic DNA (gDNA) template, which could be directly used for PCR, was developed. Cells from colonies or liquid cultures were collected and sequentially treated by microwave, plMAN5C, proteinase K and boiling (MMPB) in a single tube to give cell lysates that were qualified as PCR templates. Single-copied gDNA fragments o up to 2.8 kb were successfully amplified. We also demonstrated successful application of this method for species in the Ascomycetes and Basidiomycetes and identification of two leucine auxotroph mutants of Rhodotorula glutinis. This method could be widely employed for the screening and genetic engineering of various yeasts.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Proteinase K from Tritirachium album, ≥500 units/mL, buffered aqueous glycerol solution
Sigma-Aldrich
Proteinase K from Tritirachium album, lyophilized powder, BioUltra, ≥30 units/mg protein, for molecular biology
Sigma-Aldrich
Proteinase K from Tritirachium album, lyophilized powder, ≥30 units/mg protein
Sigma-Aldrich
Proteinase K from Tritirachium album, ≥3.0 unit/mg solid, lyophilized powder
Sigma-Aldrich
Proteinase K from Tritirachium album, buffered aqueous glycerol solution, for molecular biology, ≥800 units/mL