Abstract: Plasmid DNA from single yeast colonies was efficiently amplified using rolling circle amplification (RCA). The amplified DNA was directly used for restriction digestion, DNA sequencing, and yeast transformation. The RCA of plasmid DNA from single yeast colonies for direct retransformation of yeast simplifies conventional procedures for yeast two-hybrid analysis and is suitable for high-throughput analyses. In summary, we have developed several methods to manipulate plasmid DNA in yeast. It will greatly simplify a number of yeast-based molecular biology tools, particularly yeast two-hybrid analysis. The methods are very useful for high-throughput assays.