This protocol has been successfully used to isolate autonomous plasmids from S. cerevisiae. As a modified alkaline lysis procedure, genomic DNA is virtually eliminated from the preparation. Furthermore, the method can be easily adapted for plasmid isolation from E. coli.

Principle

E.Z.N.A^® Yeast Plasmid Isolation Kit combines the power of HiBind^® technology with the alkaline-SDS lysis of yeast cells to deliver high quality DNA in less than 1 hour. HiBind™ matrix avidly, but reversibly, binds DNA under certain optimal conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with de-ionized water or low salt buffer. For convenience, Q-Spin and V-Spin spin columns are available for centrifugation and vacuum protocols. The use of mini-columns facilitate the binding, washing, and elution steps thus enabling multiple samples to be simultaneously processed. For more information on columns, click Flexible Multi-Format Columns.

Procedure

Pellet 1.5-5 ml Yeast cells carrying desired plasmid by centrifugation and re-suspend in YP I/RNase A. Add YP II and mix gently to obtain a clear lysate. Add YP III and gently mix until a flocculent white precipitate forms. Centrifuge to pellet the cellular debris and genomic DNA. Add the clear supernatant to a clean Type I HiBind^® mini prep column and centrifuge to completely pass lysate through column. Wash the column once with Buffer HB and twice with DNA wash buffer diluted with ethanol. Centrifuge the empty column for 1 minutes to dry the column matrix. Elute the plasmid DNA with sterile de-ionized water or TE buffer.

Applications

E.Z.N.A^® Yeast Plasmid Isolation Kit provides high quality, reproducible plasmid DNA for use in the following applications :

• Automated fluorescent DNA sequencing
• Restriction endonuclease digestion

Two recombinant yeast plasmids were purified by using
the E.Z.N.A^® Yeast Plasmid Isolation Kit, and digested
with Eco RV. Digests were performed at 37° C for 1 hour
with 5 μg plasmid in each lane. M (1-kb size marker).