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Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

Kepka, Cecilia LU ; Rhodin, Jenny LU ; Lemmens, Raf; Tjerneld, Folke LU and Gustavsson, Per-Erik LU (2004) In Journal of Chromatography A 1024(1-2). p.95-104
Abstract
The primary purification of a 6.1kilo 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... (More)
The primary purification of a 6.1kilo 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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Escherichia coli, Aqueous two-phase systems, Thermoseparation, DNA
in
Journal of Chromatography A
volume
1024
issue
1-2
pages
95 - 104
publisher
Elsevier
external identifiers
  • pmid:14753711
  • wos:000187636600012
  • scopus:0348110469
ISSN
0021-9673
DOI
10.1016/j.chroma.2003.10.028
language
English
LU publication?
yes
id
f0ad27c4-a9e9-4563-84de-b2c0c023dba6 (old id 120856)
date added to LUP
2007-07-06 09:13:40
date last changed
2017-10-01 04:41:45
@article{f0ad27c4-a9e9-4563-84de-b2c0c023dba6,
  abstract     = {The primary purification of a 6.1kilo 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.},
  author       = {Kepka, Cecilia and Rhodin, Jenny and Lemmens, Raf and Tjerneld, Folke and Gustavsson, Per-Erik},
  issn         = {0021-9673},
  keyword      = {Escherichia coli,Aqueous two-phase systems,Thermoseparation,DNA},
  language     = {eng},
  number       = {1-2},
  pages        = {95--104},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system},
  url          = {http://dx.doi.org/10.1016/j.chroma.2003.10.028},
  volume       = {1024},
  year         = {2004},
}