Plasmid DNA production and purification using polyethylenimine(PEI) immobilized membrane

• Main Author: 林志仲
• Other Authors: 李振綱
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/73239129869759334703

碩士 === 國立臺灣科技大學 === 化學工程系 === 90 === Abstract Plasmid pEGFP-C1 was transformed into various Escherichia coli (E. coli) hosts: BL21 (DE3), Top 10, DH5α and JM109, respectively for evaluates a best host for plasmid production. The growth curves, plasmid content and the ratio of supercoil structure of plasmid in those transformants were studied by cultivating in LB, SDCAS and Defined mediums, respectively. E.coli JM 109 growing in Defined medium resulted in a best plasmid yield. JM 109 and defined medium were employed for plasmid production in a fermentor. The presence of Kanamycin shows a significant influence on plasmid stability. Batch fermentation by intermittent feeding Kanamycin can produce 2.98 mg/L plasmid. Polyethylenimine (PEI) is a positive polymer, which can be covalently bound on an activated microspores membrane. Using this PEI membrane can efficiently and fast recover plasmid via electrostatic interaction. About 60% of plasmid in the crude extract of 3ml cell culture can be recovered by using a single sheet (25mm diameter) of PEI membrane. The purity of the recovered plasmid is A260/280=1.8~2.1. The purity is also comparable with that brained by using BioRed mini kit as shown in agarose gel electrophoresis. The PEI membrane can be reused at least for 5 times without losing its purification capability.