Enhanced purification of plasmid DNA using Q-Sepharose by modulation of alcohol concentrations.
Ion-exchange chromatography is one of the most commonly used methods for plasmid preparation. In this study a modified method was used to purify plasmid from bacterial lysate using Q-Sepharose. Incorporation of alcohols into the washing buffers enhanced the separation of plasmid from RNA and proteins. The use of isopropanol and ethanol achieved a high yield and purity whereas the use of methanol failed to improve the plasmid purification using Q-Sepharose by batch adsorption-desorption. Stepwise elution containing various concentrations of isopropanol and NaCl was used in preparative chromatography to enhance the plasmid purification. The same stepwise elution was applied to the chromatography columns packed with 0.5, 20, and 200 ml of Q-Sepharose for plasmid purification from 7.5, 300, and 3000 ml bacterial broth, respectively. Complete separation of DNA from RNA and proteins was achieved under gravity flow by modulation of the alcohol concentrations in the stepwise elution. These three scales of chromatography maintained an approximate plasmid yield and the purified plasmid contained undetectable levels of RNA and protein.
Tseng
Wen-Chi
WC
Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Rd., Taipei 106, Taiwan. tsengwc@ch.ntust.edu.tw
Ho
Fu-Lyn
FL
D019840:2-Propanol / Q000737:chemistry*; D000327:Adsorption; D002021:Buffers; D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006112:Gravitation; D010957:Plasmids; D012685:Sepharose / Q000737:chemistry*
D002021:Buffers; D004247:DNA; D012685:Sepharose; D019840:2-Propanol