Purification of plasmid DNA by fast protein liquid chromatography on superose 6 preparative grade.
We were able to reduce both the time and the use of hazardous chemicals associated with the previous plasmid isolation methods of high-pressure liquid chromatography and CsCl gradient centrifugation by employing fast protein liquid chromatography (FPLC). Plasmid was first crudely prepared from bacterial cultures by a standard alkaline lysis method. After an alcohol precipitation, the nucleic acids were divided into two equal portions. One half was used for a standard purification method employing CsCl centrifugation. The other was dissolved in FPLC buffer, treated with RNase A, and applied to a Superose 6 preparative grade column (HR 10/30). Plasmid eluted off the column within 20 min as a single, highly resolved peak. Plasmid isolated by FPLC had yields, purity, and transformation efficiencies similar to that isolated by CsCl centrifugation.
McClung
J K
JK
Samuel Roberts Noble Foundation, Inc., Biomedical Division, Ardmore, Oklahoma 73402.
Gonzales
R A
RA
D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics; D012685:Sepharose
D004269:DNA, Bacterial; D012685:Sepharose