Related Experiment Video
Updated: Apr 18, 2026
06:53
Streamlined Purification of Plasmid DNA From Prokaryotic Cultures
Published on: January 5, 2011
20.6K
Plasmid DNA primary recovery from E. coli lysates by depth bed microfiltration.
Adriana Padilla-Zamudio1, Patricia Guerrero-Germán, Armando Tejeda-Mansir
1Department of Chemical Engineering and Metallurgy, University of Sonora, Blvd. Luis Encinas s/n, 83000, Hermosillo, Sonora, Mexico.
Bioprocess and Biosystems Engineering
|January 13, 2015
Summary
Depth bed microfiltration effectively clarifies bacterial lysates for plasmid DNA (pDNA) production, removing 99% of solids. This method offers a competitive alternative to traditional centrifugation in bioprocessing.
Area of Science:
- Biotechnology
- Bioprocess Engineering
- Molecular Biology
Background:
- Plasmid DNA (pDNA) is crucial for gene therapy and vaccine development.
- pDNA production involves fermentation, recovery, and purification steps.
- Primary recovery, including lysate clarification, is a critical step.
Purpose of the Study:
- To investigate depth bed microfiltration for clarifying E. coli lysates containing pVAX1-NH36 plasmid DNA.
- To compare the efficacy of microfiltration with conventional centrifugation for lysate clarification.
Main Methods:
- Utilized depth bed microfiltration with 8.0 µm pore size filters.
- Tested fluxes of 0.0027 and 0.004 cm³/cm²/s.
- Employed a fiber coating model to analyze the microfiltration system's behavior.
Main Results:
- Achieved 99% solids elimination from cell lysates using depth bed microfiltration.
- Solids occupied 23% and 43% of the void volume at different flow rates, indicating early breakthrough.
- Depth bed microfiltration demonstrated competitive results compared to centrifugation.
Conclusions:
- Depth bed microfiltration is an effective method for clarifying bacterial lysates in pDNA production.
- The process shows promise as an alternative to centrifugation for primary recovery.
- Further optimization may enhance efficiency and reduce breakthrough issues.
Related Concept Videos
01:24DNA Isolation
47.2K
DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
47.2K
02:35DNA Agarose Gel Electrophoresis
122.5K
Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify DNA fragments using a gel extraction protocol.
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA...
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA...
122.5K