Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure

Yeast - Tập 11 Số 4 - Trang 355-360 - 1995
R. Daniel Gietz1, Robert H. Schiestl2, Andrew Willems3, Robin A. Woods4,2
1Department of Human Genetics, University of Manitoba, Winnipeg, Canada
2Department of Molecular and Cellular Toxicology, Harvard School of Public Health, 665 Huntington Ave, Boston, Massachusetts 02115, U.S.A.
3Dept of Biology, University of Winnipeg, 515 Portage Avenue, Winnipeg, Manitoba, Canada R3B 2E9
4Department of Biology, University of Winnipeg, 515 Portage Avenue, Winnipeg, Manitoba, Canada R3B 2E9

Tóm tắt

Abstract

An improved lithium acetate (LiAc)/single‐stranded DNA (SS‐DNA)/polyethylene glycol (PEG) protocol which yields >1 × 106 transformants/μg plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS‐DNA/PEG transformation. The highest transformation efficiency was observed when 1 × 108 cells were transformed with 100 ng plasmid DNA in the presence of 50 μg SS carrier DNA. The yield of transformants increased linearly up to 5 μg plasmid per transformation. A 20‐min heat shock at 42°C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P‐labelled plasmid DNA than did double‐stranded (DS) carrier. The LiAc/SS‐DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS‐DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS‐DNA/PEG method is discussed.

Từ khóa


Tài liệu tham khảo

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Yeast

Tập 11 Số 4

355-360

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