Plasmid DNA Purity Grades

Transfection-grade plasmid purification kits

Molecular-grade plasmid isolation technologies are generally faster, more cost-effective and are best suited for robust applications such as cloning, nucleic acid labeling, PCR, and sequencing, where high purity levels are not required. Our GeneJET plasmid DNA purification kits are best suited for molecular-grade plasmid DNA isolation.

Transfection-grade plasmid purification technologies may be used for applications that demand greater purity and higher yields than molecular-grade plasmid DNA. Our PureLink HiPure plasmid purification kits and PureLink Fast Low Endotoxin plasmid purification kits produce the higher yields and lower endotoxin levels required for more sensitive applications, such as transfection of standard cell lines and in vitro transcription. Transfection-grade plasmid DNA is also suitable for all molecular-grade applications, such as cloning or sequencing.

Advanced transfection-grade plasmid isolation technology is suited for the most sensitive applications, but can also be used for all molecular and transfection-grade applications. Our PureLink Expi Endotoxin-Free plasmid purification kits offer rapid purification of large-scale, endotoxin-free (<0.1 EU/µg) plasmid that is ideal for transfection of sensitive cell lines (i.e., primary cells) and experiments in vivo (research on gene therapy, microinjections, and vaccines).

Choose the right transfection reagent for your desired sample type and workflow.

Downstream application and purity grade requirements

Downstream application	Purity grade
Downstream application	Molecular	Transfection	Advanced transfection
	GeneJET kits	PureLink HiPure kits and PureLink Fast Low Endotoxin kits	PureLink Expi Endotoxin-Free kits
Sensitive transfection (i.e., primary cells)
Vaccine research
Microinjection
Gene therapy research
In vitro transcription
Standard transfection
Cloning
Nucleic acid labeling
PCR
Sequencing
Transformation

The reality of endotoxin levels and transfection efficiency

The Myth		Endotoxins in purified plasmid DNA can decrease transfection efficiency and viability in all cell lines and requires “endotoxin-free” (<0.1 EU/µg) plasmid purification.
The Scientific Reality		Only high levels of endotoxin (>10 EU/µg) have been shown to negatively impact transfection, protein expression, and viability in standard cell lines. Sensitive applications, such as transfection of primary or stem cells, and other in vivo applications require endotoxin-free (<0.1 EU/µg) plasmid DNA.¹
Our Solution		Low levels of endotoxin (0.1 – 10 EU/µg) produced by anion exchange plasmid purification systems, such as PureLink HiPure and PureLink Fast Low Endotoxin kits, are ideal for the majority of transfection applications. For the most sensitive applications, advanced transfection-grade endotoxin-free plasmid DNA (<0.1 EU/µg) is recommended.

Plasmid myths and facts!

Myth: Endotoxins in purified plasmid DNA can decrease transfection efficiency and viability in all cell lines and require costly “endotoxin-free” (2,000 EU/μg).

Fact: Only dramatically high levels of endotoxin (>2,000 EU/μg) have been shown to negatively impact transfection and protein expression in standard cell lines.¹

Myth: Plasmid purification is not affected by RNA contamination.

Fact: Despite the addition of RNase A in plasmid purification kits if using silica resin-based kits, bacterial RNA contamination can be an issue.

Myth: The RNA present in my plasmid DNA is not a concern; it will not affect my workflow.

Fact: When used in sensitive downstream applications such as transfection, high levels of RNA can lead to suboptimal expression, cell toxicity, or activation of confounding signaling pathways.

Myth: RNA contamination is not affected by purification resin.

Fact: Anion exchange resin-based plasmid purification kits produce ultralow RNA contamination ideal for transfection, whereas silica-based kits produce relatively high levels of RNA contamination.

Myth: I can quantitate my plasmid DNA accurately with a spectrophotometer.

Fact: Standard cuvette-based spectrophotometers cannot accurately differentiate RNA from plasmid DNA, which leads to overestimation of the total plasmid yield.

Endotoxin values can vary with bacterial strain, plasmid backbone, and bacterial pellet size. Low endotoxin is ideal for the majority of transfection experiments.¹

¹Butash, KA et al. (2000) Reexamination of the Effect of Endotoxin on Cell Proliferation and Transfection Efficiency. Biotechniques 29(3): 610-614, 616, 618-619.

Maximize plasmid DNA transfection results

Success in transfection relies on high transfection efficiency and low cytotoxicity. Transfection efficiency is a measure of the transport of a molecule―in this case plasmid DNA—into a cell. This efficiency is influenced by many different conditions, including the quality of the plasmid DNA.

Plasmid DNA purified in a single pass with PureLink HiPure Plasmid and PureLink Fast Low Endotoxin kits supports high transfection efficiencies with the low cytotoxicity seen with DNA prepared with other high-purity, anion exchange-based purification kits. According to research done in 2000, cell lines used for transfection studies do not require endotoxin-free DNA. In fact, only endotoxin levels of at least 10,000 EUs significantly affect cell proliferation and viability.

Cell lines compatible with PureLink HiPure and PureLink Fast Low Endotoxin kits
HeLa HEK293 HEK293T NIH3T3	CHO-S Jurkat CHO-K1 K562	Huh-7 COS-7L PC-12

What is endotoxin or lipopolysaccharide (LPS)?

Endotoxin, also known as lipopolysaccharide or LPS, is a component of the plasma membrane of gram-negative bacteria such as E. coli (Figure 1), and a common contaminant in plasmid preparations. Endotoxin levels are commonly reported as endotoxin units per microgram of plasmid DNA (EU/µg DNA). High amounts of endotoxins are released from the lysed bacteria during the plasmid purification process and these molecules tend to co-purify with plasmid DNA due to similar chemical properties. Depending on the downstream application for the plasmid DNA, researchers may want to utilize specialized kits to minimize endotoxin levels in their preparations.

Figure 1. Diagram of the gram-negative bacterial membrane composition. Source: Kenneth Todar, at the University of Wisconsin-Madison Department of Bacteriology, On-line textbook of Bacteriology.

Effect of endotoxin levels on transfection, cell viability, and gene expression

Depending on the downstream application, endotoxins in plasmid preparations can negatively impact the outcome of the experiments. Endotoxins affect the interaction between the transfection reagent and plasmid, thereby reducing efficiency of the plasmid delivery and gene expression. Some cell lines are sensitive to endotoxin and depending on the level of endotoxins, their viability will be negatively affected, and intracellular biology will be significantly altered. Additionally, endotoxin levels can activate unwanted immune responses in cell culture or animal experiments.

How to remove endotoxins during plasmid purification

We offer a range of plasmid isolation kits with different amounts of endotoxin depletion appropriate for your application. These kits utilize various proprietary resins and buffers that produce plasmid suitable for molecular biology applications (>10 EU/µg), standard transfection (0.1 – 1.0 EU/µg, “low endotoxin”), and advanced transfection (<0.1 EU/µg, “endotoxin-free”) applications. The silica-based resin in the GeneJET Plasmid Purification kits enables purification of plasmids with >10 EU/µg, which is suitable for molecular biology applications, such as sequencing, cloning, and PCR. PureLink HiPure and PureLink Fast Low Endotoxin kits enable purification of plasmid DNA with low levels of endotoxins (0.1 – 1.0 EU/µg), suitable for standard transfection applications. The PureLink Expi Endotoxin-Free kits utilize novel anion exchange membranes, as well as a proprietary endotoxin removal buffer and endotoxin-free components for rapid production of endotoxin-free (<0.1 EU/µg) plasmid purifications enabling any downstream application, including transfection into sensitive cell lines and in vivo experiments.

Endotoxins can be measured using two methods

One method to measure endotoxin levels involves measuring a clotting reaction between the endotoxin and a clottable protein in the amoebocytes of Limulus polyphemus, the horseshoe crab. A much more sensitive photometric test is based on a Limulus amoebocyte lysate (LAL) and a synthetic color-producing substrate. This LAL assay is used for the routine analysis of endotoxin levels in biological solutions and was the first detection method to be certified by the FDA.

Endotoxin levels

The reality of endotoxin levels and transfection efficiency

The Myth		Endotoxins in purified plasmid DNA can decrease transfection efficiency and viability in all cell lines and requires “endotoxin-free” (<0.1 EU/µg) plasmid purification.
The Scientific Reality		Only high levels of endotoxin (>10 EU/µg) have been shown to negatively impact transfection, protein expression, and viability in standard cell lines. Sensitive applications, such as transfection of primary or stem cells, and other in vivo applications require endotoxin-free (<0.1 EU/µg) plasmid DNA.¹
Our Solution		Low levels of endotoxin (0.1 – 10 EU/µg) produced by anion exchange plasmid purification systems, such as PureLink HiPure and PureLink Fast Low Endotoxin kits, are ideal for the majority of transfection applications. For the most sensitive applications, advanced transfection-grade endotoxin-free plasmid DNA (<0.1 EU/µg) is recommended.