Plasmid isolated with traditional purification procedures normally contain high levels of endotoxins (also known as lipopolysaccharides or LPS) that can significantly interfere with transfection experiments downstream. Up to 5 mL of overnight cultures can be used for high copy number plasmids and 10 mL for low copy number. The E.Z.N.A. Endo-Free Plasmid Mini Kit I integrates an efficient endotoxin removal step into the plasmid purification procedure to produce high-quality transfection grade (<0.1 EU/µg) plasmid for efficient transfection. The bacterial cells are lysed using the alkaline-SDS lysis method. The cleared cell lysate is then treated with ETR reagent to efficiently remove the endotoxins. After adjusting the binding condition, the cell lysate is applied into the HiBind DNA column and purified DNA is eluted from the column membrane.