How To Concentrate Plasmid DNA

BioInnovatise Plasmid DNA Team

Updated May 28, 2024

Plasmid DNA Basics

Plasmids, or pDNA, are usually circular molecules of DNA¹. Plasmids can only multiply inside a host cell such as bacteria. E. Coli bacteria is a suitable host cell for plasmid DNA because of its fast-growing nature, ease to manipulate genetically, and has well-characterized genetics

Most plasmids inhabit bacteria, and around 50% of bacteria found in the wild contain one or more plasmids. Plasmid DNA can be manufactured using host cells from mammalian cells (frequently used in cell and gene therapy research), insect cells (baculovirus or SF9), plant cells, or yeast cells for eukaryotic cell research.

“The copy number is the number of copies of the plasmid in each bacterial cell. For most plasmids, it is 1 or 2 copies per chromosome, but it may be as many as 50 or more for certain small plasmids such as the ColE plasmids. The number of copies influences the strength of plasmid-borne characteristics, especially antibiotic resistance. The more copies of the plasmid per cell, the more copies there will be of the antibiotic resistance genes, and therefore, the higher the resulting level of antibiotic resistance.” ScienceDirect.

How To Concentrate Plasmid DNA

Concentrating plasmid DNA is often included in most plasmid DNA preparation protocols. Plasmid preparation, known as plasmid prep typically involves several key steps:

Cell Lysis: Breaking open bacterial cells to release plasmid DNA.
Clearing Lysate: Removing cell debris, proteins, and other contaminants.
Binding DNA: Using silica columns or other materials to bind plasmid DNA while washing away impurities.
Washing: Further purification to ensure removal of salts and other contaminants.
Eluting: Releasing purified plasmid DNA from the binding material.
Concentrating: Increasing the plasmid DNA concentration for specific applications.

Concentrating plasmid DNA in a laboratory setting can be achieved through several methods, depending on the volume such as plasmid midiprep, plasmid maxiprep, plasmid megaprep, and the purity required (a 1.8 260/280 nucleic acid ratio is considered a pure enough concentration of plasmid DNA for research applications). These are some of the ways how plasmid DNA is concentrated:

Ethanol or Isopropanol
Precipitation
Spin Column
Vacuum Concentration
Ultrafiltration
PEG Precipitation

Our laboratory manufactures and prepares plasmid DNA using a variety of techniques. If you have a specific technique required for your plasmid DNA application, let us know when requesting a production.

The above diagram illustrates the plasmid prep production process at BioInnovatise which produces endotoxin free and animal component free plasmid DNA. We perform plasmid prep using our proprietary plasmid DNA preparation protocol.

How to Measure The Concentration of Plasmid DNA

Each method of concentrating plasmid DNA has its own advantages and is suitable for different applications. Our team has prepared a list of the more common measurement techniques for measuring plasmid DNA concentration:

Spectrophotometry (UV Absorbance)
- Principle: DNA absorbs ultraviolet (UV) light at a wavelength of 260 nm. The absorbance at this wavelength can be used to calculate the concentration of DNA.
- Purity Check: Measure absorbance at 280 nm (A280) to assess protein contamination. The A260/A280 ratio should be between 1.8 and 2.0 for pure DNA.
- Advantages: Quick, easy, and non-destructive.
- Limitations: Requires relatively clean DNA samples; contaminants can affect accuracy.
Fluorometry
- Principle: Fluorescent dyes that specifically bind to DNA (e.g., PicoGreen, Qubit dsDNA BR Assay) emit fluorescence upon binding, which can be quantified.
- Advantages: Highly sensitive and specific; less affected by contaminants compared to spectrophotometry.
- Limitations: Requires specific dyes and equipment.
Agarose Gel Electrophoresis
- Principle: DNA is separated by size on an agarose gel and visualized using a DNA-binding dye (e.g., ethidium bromide or SYBR Safe). The intensity of the DNA bands can be compared to a standard.
- Advantages: Provides visual confirmation of DNA size and integrity.
- Limitations: Semi-quantitative; less precise than spectrophotometry or fluorometry.
Nanodrop Spectrophotometer
- Principle: A specialized type of spectrophotometer that measures absorbance at 260 nm and 280 nm with very small sample volumes (1-2 µL).
- Advantages: Quick, requires minimal sample, and provides both concentration and purity information.
- Limitations: Accuracy can be affected by contaminants.

Why Is the Concentration of Plasmid DNA Important

Not only is the concentration of plasmid DNA important, but so is the accurate measurement. If you have an inaccurate measurement of plasmid DNA, it will have large consequences on your downstream applications.

Because endotoxin free plasmid DNA is important for downstream cell and gene therapy, immunology, oncology, regenerative medicine, and vaccine development research applications, the concentration of plasmid DNA plays an important role. Here are some of the reasons why plasmid DNA concentration is important:

Efficiency in Downstream Applications
- Transformation Efficiency: Higher DNA concentrations increase the likelihood of successful bacterial transformation or transfection of eukaryotic cells. This is important for cloning or mutagenesis, gene expression studies, and mutation studies.
- Sequencing: DNA sequencing, especially next-generation sequencing (NGS), often requires precise DNA concentrations for optimal library preparation and sequencing accuracy.
Optimizing Reagent Use
- Cost Efficiency: Using the correct DNA concentration prevents waste of reagents, such as enzymes, primers, and other components, thereby reducing experimental costs.
- Reaction Efficiency: High concentrations can lead to inhibition or non-specific binding in enzymatic reactions, while low concentrations might result in insufficient substrate for reactions, both of which can affect experimental outcomes.
Vector Copy Number
- Expression Levels: In gene expression studies, the concentration of plasmid DNA can influence the copy number of the vector in host cells, directly affecting the level of gene expression. High copy number plasmids can lead to higher protein yields.
- Toxicity and Burden: Overexpression of certain genes can be toxic to host cells. Maintaining an optimal plasmid DNA concentration helps balance expression levels to avoid cellular stress or death.
Gene Therapy and Vaccine Development
- Dose Accuracy: In gene therapy, accurate dosing of plasmid DNA is critical to ensure therapeutic efficacy and safety. Too much DNA can lead to adverse immune responses, while too little might not produce the desired therapeutic effect. Learn more about our lentivirus packaging and retrovirus packaging services.
- Regulatory Compliance: For clinical applications, regulatory agencies require precise quantification of DNA used in therapies and vaccines to ensure consistent dosing and efficacy.

How Can I Start A Plasmid DNA Preparation Production

Let’s get started! Our plasmid team is excited to bring your project to life. In order to get started please provide following when requesting a production:

Plasmid copy number
Specification of antibiotic resistance of the plasmid
1 μg of wildtype plasmid

Learn more about our quick turnaround endotoxin free plasmid DNA prep services.

Precision medicine research and development progresses everyday, and with it, the need for high quality plasmid DNA.

Want to learn more about the latest in plasmid research? Our colleagues at Science Direct and the American Society for Biochemistry and Molecular Biology are always collecting and publishing the latest information and research.

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