[Construction of Streptococcus mutans surface protein antigen eukaryotic expression vector pcDNA3-PAc. I. Extraction and purification of plasmid DNA pPC41 and pcDNA3].
OBJECTIVE
To Extract and purify plasmid DNA pPC41 and pcDNA3.


METHODS
Bacteria were collected and lyzed by the alkaline lysis method. The plasmid pPC41 carrying structural gene pac and clone expression vector pcDNA3 were extracted and purified from E. coli clone by the polyethylene glycol precipitating method, the electroelution into dialysis bags method, the low melting-temperature agarose gel and glass fiber column chromatography. The concentration, purity and molecular weight were determined by spectrophotometry, restriction enzyme and agarose gel electrophoresis.


RESULTS
The concentration of plasmid DNA of this experiment in general was 0.12-0.24 g/L, and A260/A280 of the polyethylene glycol precipitating method, the electroelution into dialysis bags method, the low metling-temperature agrose gel and glass fiber column chromatography were respectively 1.9, 2.2, 2.2 and 2.6. Molecular weight of pPC41 and pcDNA3 was respectively 10.6 kb and 5.4 kb.


CONCLUSIONS
The four methods can obtain pure plasmid DNA. Purity of plasmid DNA obtained by glass fiber column chromatography method is the highest in the four methods. Glass fiber column chromatography is one of the effective methods getting highly purified plasmid DNA.
Liu
J
J
College of Stomatology, West China University of Medical Sciences.
Liu
T
T
Zhou
X
X
Liu
L
L
Jia
W
W
Zhan
L
L
D000954:Antigens, Surface; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics*; D013295:Streptococcus mutans / Q000235:genetics*
D000954:Antigens, Surface; D004269:DNA, Bacterial