High performance DNA purification using a novel ion exchange matrix.
Ion exchange chromatography has emerged as a reliable alternative to classic CsCl-ethidium bromide gradients for isolating nucleic acids of the highest purity. A plasmid purification method based on a unique anion exchange membrane (IEXM) was developed for the production of superior quality plasmids. This method was simpler and more efficient than conventional bead-based methods. Plasmids were extracted from bacterial cells through alkaline lysis. The crude lysate was clarified by a sequential filtration device that not only removed cell debris but micellar aggregates as well. The clarified lysate was mixed with an extraction solution and loaded into a spin column containing IEXM. Binding, washing, and elution conditions were optimized to achieve efficient isolation of plasmids from the impurities. IEXM had an exceedingly high dynamic binding capacity, excellent selectivity, and a near 100% recovery for plasmids. The binding capacity for pUC19 was 2.93 mg/cm(3) of IEXM, which is several times greater than the values for conventional ion exchange beads. The superior selectivity of the method was reflected in the extremely low levels of endotoxin, and thus it is well-suited for critical applications in eukaryotic systems.
Yang
Yu
Y
EdgeBio, Gaithersburg, MD 20877, USA.
Hebron
Haroun R
HR
Hang
Jun
J
D001709:Biotechnology; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D004731:Endotoxins / Q000302:isolation & purification; D007474:Ion Exchange; D008567:Membranes, Artificial; D010957:Plasmids / Q000302:isolation & purification; D013053:Spectrophotometry
D004731:Endotoxins; D008567:Membranes, Artificial; D004247:DNA