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 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).
|Downstream application||Purity grade|
|GeneJET kits||PureLink HiPure kits||PureLink Endotoxin-Free kits|
|Sensitive transfection (i.e., primary cells)||✓|
|Gene therapy research||✓|
In vitro transcription
|Nucleic acid labeling||✓||✓||✓|
The reality of endotoxin levels and transfection efficiency
||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.1 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
Low levels of endotoxin (0.1 – 10 EU/µg) produced by anion exchange plasmid purification systems, such as PureLink HiPure 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.
Endotoxin values can vary with bacteria strain, plasmid backbone, and bacterial pellet size. Low endotoxin is ideal for the majority of transfection experiments.1
Maximizing 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 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 Kits|
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 effect 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. The anion exchange resin in the PureLink HiPure kits enables 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.
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