Affinity purification of plasmid DNA directly from crude bacterial cell lysates
| Autor: | Nigel K. H. Slater, Gareth M. Forde, Anna V. Hine, Richard A. J. Darby |
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| Rok vydání: | 2007 |
| Předmět: |
Isopropyl Thiogalactoside
Recombinant Fusion Proteins Green Fluorescent Proteins Bioengineering Biology Applied Microbiology and Biotechnology Chromatography Affinity chemistry.chemical_compound Plasmid Bacterial Proteins Affinity chromatography Escherichia coli Lac Repressors Histidine Protein–DNA interaction Plasmid preparation Aspartic Acid Chromatography Bacteria DNA Superhelical Escherichia coli Proteins Sepharose Reproducibility of Results Ribonuclease Pancreatic DNA extraction Repressor Proteins genomic DNA Freeze Drying chemistry DNA supercoil DNA Plasmids Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 98:1103-1108 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.21492 |
| Popis: | We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required. © 2007 Wiley Periodicals, Inc. |
| Databáze: | OpenAIRE |
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