Our Yeast Plasmid MiniPrep Kit is designed for rapid and cost-effective small-scale preparation of high quality yeast plasmid DNA. The method is based on a silica- membrane spin column.This method requires few manipulations, and is both faster and easier to perform than other organic-based extraction methods. The purified DNA is suitable for all common molecular biology procedures, including restriction digestion, cloning, sequencing, etc.
Principle
Due to the presence of a more resistant cell wall than the one present in E.coli, the plasmid DNA isolation methods commonly used for the yeast Saccharomyces cerevisiae are more difficult and produce lower DNA yields than bacterial plasmid isolation methods.
In our protocol, we perform an initial step to remove the exterior wall from yeast in order to obtain yeast protoplasts. Starting from protoplasts, we use the Yeast Plasmid DNA Purification kit to isolate plasmid DNA from yeast.
First we use lyticase to digest the wall of the yeast to obtain yeast protoplasts. Pelleted protoplasts are resuspended and subjected to SDS/alkaline lysis to liberate the plasmid DNA. The resulting lysate is neutralized to create appropriate conditions for binding of plasmid DNA on the silica membrane in the spin column . Cell debris and SDS precipitate are pelleted by centrifugation, and the supernatant containing the plasmid DNA is loaded onto the spin column membrane. The adsorbed DNA is washed to remove contaminants, and is then eluted with a small volume of the Elution Buffer (10 mM Tris-HCl, pH 8.5).The purified plasmid DNA is ready for immediate use in all molecular biology procedures such as conventional digestion with restriction enzymes, PCR, transformation and automated sequencing.
Features
High purity: A260/A280=1.7-1.9. with no chloroform and pheno.
