Protocol Online logo
Top : Forum Archives: : Molecular Biology

purifying supercoiled DNA? - trying to separate supercoiled from nicked (Jul/09/2002 )

Does anyone know of a good method for separating supercoiled plasmid DNA from nicked?  I have tried sucrose density gradient centrifugation with little luck -- and I know there has to be an easier way!  


-Chris Ingle-

Simple method is to make again alkaline lysis and neutralization by acid kalium acetate in low volume (300 ul/100 ug of DNA) and freeze solution after neutralization. Open cicrcle and linear form of plasmide renaturates randomly and accumulates in sediment. Supernatant containing supercoil can be precipitated by ethanol or isolated on Qiagen column. Method is not absolute, but enrichment of supercoil is substantial.