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African Journal of Biotechnology

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The technology of large-scale pharmaceutical plasmid purification by cetyltrimethylammonium bromide and Tritonx-114

Q Zhang, WF Yuan, HC Sun, SH Hou, HF Zhu

Abstract


Many methods for plasmid purification have been developed, and the whole process must be designed to remove the host RNA, protein, genomic DNA and endotoxin. Currently, plasmid is mostly purified by time-consuming chromatographies. As an alternative, a new plasmid purification technology with cetyltrimethylammonium bromide (CTAB) is described. After lysis with alkali, the CTAB was directly titrated into the supernatant for plasmid precipitation, then the coprecipitated pellets were dealt with 3 M KAc and TritonX-114. Quality detection showed that the purified plasmids were free from the contamination of host RNA. In 1 mg purified plasmid, the bacterial genomic DNA, host endotoxin and protein were less than 10 g/ mg, 50 EU/ mg and 10 g/mg, respectively. The ratio of OD260/OD280 was between 1.75 - 1.85, more than 90% of the prepared plasmid presented in the supercoiled form. Further test demonstrated that the pcDNAlacZ purified with CTAB and authoritative endotoxin-free plasmid Kit had the similar transfection efficiency in vivo and in vitro. CTAB can be used for plasmid purification; the main advantages of the DNAs purified with CTAB include the avoidance of animal-derived enzymes, toxic substance like chloroform and phenol. More attractive is that the whole process has the predominance of low cost.



AJOL African Journals Online