Development of a New Method for Rapid Purification and Analysis of Circular Plasmid DNAs from Yeast Cells
Date
2024-04
Authors
Ali, Ahmed Wajahat
Sowersby, Drew S.
Okoye, Linda C.
Lewis, L. Kevin
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Abstract
It is common practice to purify circular plasmid DNAs from small cultures of E. coli bacterial cells for subsequent analysis by gel electrophoresis. By contrast, extraction and electrophoretic analysis of circular DNAs from small cultures of yeast and other eukaryotic cells is difficult because of the small numbers of these molecules inside cells. Existing methods employed in budding yeast (S. cerevisiae) require extraction from a large volume of cultured cells; the partially purified plasmids are then typically transformed into E. coli cells and extracted via plasmid DNA minipreps for gel analysis. In the current study, we have developed a new method for extracting plasmids from small yeast cell cultures that permits visualization of the circular DNAs by electrophoresis. The new approach was shown to be superior to two other common yeast DNA extraction methods, as it produced more plasmid DNA and less contaminating chromosomal DNA and RNA. The method was tested by cloning a RAD52 gene-containing fragment into an expression vector inside yeast cells. DNAs from the transformants were then analyzed directly by gel electrophoresis after performing yeast minipreps. The new method allows yeast plasmids to be analyzed quickly, eliminating the requirement for subsequent transformation into E. coli cells.
Description
Keywords
gel electrophoresis, yeast circular DNA, visualization, fast
Citation
Ahmed, S., Sowersby, D. S., Okoye, L. C., & Lewis, L. K. (2024). Development of a new method for rapid purification and analysis of circular plasmid DNAs from yeast cells. Poster presented at the Graduate Student Research Conference, San Marcos, Texas.