Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors.
This paper describes a new method of plasmid DNA purification which is fast and reliable enough for most purposes in recombinant DNA technology. The present method does not require the use of toxic chemicals such as phenol or ethidium bromide, costly ultracentrifugation procedures or other processes which can modify the supercoiled structure of the plasmids, such as adsorption on glass fiber. This method is based on the principle of gel filtration chromatography, at low pressure (1 bar) or medium pressure (between 5 and 10 bars), using Sephacryl S1000 or Superose 6B. It permits recovery of plasmids: in preparative quantities (from 300 micrograms to 4 mg), exempt from RNA, DNA and protein contamination, and suitable for various common genetic engineering procedures immediately after purification. To test the reliability of the technique as well as the degree of purification, the plasmids were used to construct thermoamplifiable vectors, carrying the lacUV5 promoter and the 5' end of the beta-galactosidase gene with a single EcoR1 site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins in Escherichia coli.
Vo-Quang
T
T
Malpiece
Y
Y
Buffard
D
D
Kaminski
P A
PA
Vidal
D
D
Strosberg
A D
AD
D002850:Chromatography, Gel; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004279:DNA, Viral / Q000302:isolation & purification*; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli; D005786:Gene Expression Regulation; D007763:Lac Operon; D008722:Methods; D010957:Plasmids; D001616:beta-Galactosidase / Q000235:genetics
D004279:DNA, Viral; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D001616:beta-Galactosidase