[Purification of large-scale plasmid DNAs by selective precipitation with cetyltrimethylammonium bromide].
Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 microg, 50 EU and 10 microg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75-1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Zhang
Quan
Q
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China.
Yu
Kexiang
K
Yuan
Weifeng
W
Xue
Fangming
F
Sun
Huaichang
H
Zhu
Hongfei
H
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry*; D011232:Chemical Precipitation; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*
D002593:Cetrimonium Compounds; D004269:DNA, Bacterial; D004247:DNA; D000077286:Cetrimonium