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Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli

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Abstract

The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.

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Acknowledgments

The authors acknowledge support of their work through various grants from the Slovenian Research Agency. Research was conducted within the scope of the EBAM European Associated Laboratory (LEA). This research was possible as a result of networking efforts within COST Action TD1104 (www.electroporation.net).

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Authors and Affiliations

  1. Faculty of Electrical Engineering, Laboratory of Biocybernetics, University of Ljubljana, Trzaska 25, 1000, Ljubljana, Slovenia

    Saša Haberl, Duša Hodžić & Damijan Miklavčič

  2. BIA Separations, d.o.o, Mirce 21, 5270, Ajdovscina, Slovenia

    Marko Jarc, Aleš Štrancar & Matjaž Peterka

Authors
  1. Saša Haberl
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  2. Marko Jarc
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  3. Aleš Štrancar
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  4. Matjaž Peterka
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  5. Duša Hodžić
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  6. Damijan Miklavčič
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Correspondence to Damijan Miklavčič.

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Haberl, S., Jarc, M., Štrancar, A. et al. Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli . J Membrane Biol 246, 861–867 (2013). https://doi.org/10.1007/s00232-013-9580-5

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  • DOI: https://doi.org/10.1007/s00232-013-9580-5

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