Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system.
The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA.
Kepka
Cecilia
C
The Swedish Center for Bioseparation.
Rhodin
Jenny
J
Lemmens
Raf
R
Tjerneld
Folke
F
Gustavsson
Per-Erik c
PE
D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids / Q000302:isolation & purification*
D004247:DNA