Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/120856
- author
- Kepka, Cecilia LU ; Rhodin, Jenny LU ; Lemmens, Raf ; Tjerneld, Folke LU and Gustavsson, Per-Erik LU
- organization
- publishing date
- 2004
- 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
- 2016-04-01 16:09:46
- date last changed
- 2022-04-15 02:32:47
@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}}, keywords = {{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}}, doi = {{10.1016/j.chroma.2003.10.028}}, volume = {{1024}}, year = {{2004}}, }