Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration.
We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.
Kondo
T
T
Department of Physical Biochemistry, Gunma University, Maebashi, Japan.
Mukai
M
M
Kondo
Y
Y
D011232:Chemical Precipitation; D002712:Chlorides; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D008094:Lithium; D018021:Lithium Chloride; D010957:Plasmids
D002712:Chlorides; D004247:DNA; D008094:Lithium; D004996:Ethidium; D018021:Lithium Chloride