Molecular-grade plasmid isolation technologies are generally faster, more cost-effective and are best suited for applications such as cloning, nucleic acid labeling, PCR, and sequencing where endotoxin levels are less of a concern.
Transfection-grade plasmid purification kits may be used for all applications, from transformation to in vitro transcription. Our PureLink® HiPure plasmid purification kits produce the higher yields and lower endotoxin levels required for more sensitive applications, such as cell culture transfections, microinjection, restriction analysis, sequencing, and gene therapy.
|Downstream application||Purity grade|
|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 costly “endotoxin free” (< 0.1 EU/ µg) plasmid purification.|
|The Scientific Reality
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
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.
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.
Low endotoxin levels (ideal for transfection and cell viability) when plasmid DNA is purified using PureLink® HiPure.
Low cytotoxicity of plasmid DNA purified with the PureLink® HiPure Plasmid Kits. CHO-K1 cells were grown in 96-well plates to 80% confluence, then transfected with 200 ng/well of pEF4-luciferase reporter plasmid, using 0.1 μg increments (up to 1.0 μg) of Lipofectamine™ 2000 Transfection Reagent. At 24 hr post-transfection, the cells were treated with 10 μM camptothecin for 1 hr, then assayed using the fluorescence-based Vybrant® Cytotoxicity Assay Kit. Results for an untreated control sample (“Cells”) and cells transfected with plasmid DNA only (no transfection reagent added) are also shown. PL: plasmid DNA purified using the PureLink® HiPure Plasmid Filter Maxiprep Kit. Q: plasmid purified using the Qiagen EndoFree® Plasmid Maxi Kit.
|Cell Lines Compatible with PureLink® HiPure Kits|
All endotoxin testing for the Purelink® HiPure Kits was performed by an independent FDA-qualified third party testing facility.
Endotoxins, also known as lipopolysaccharides (LPS), are part of the outer membrane of the cell wall of Gram-negative bacteria.
Endotoxin is invariably associated with Gram-negative bacteria, regardless of whether the organisms are pathogenic. Although the term is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is properly reserved to refer to this lipopolysaccharide complex. Gram-negative bacteria containing these endotoxins include Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus influenzae, Bordetella pertussis, and Vibrio cholerae (Figure 1).
—Kenneth Todar, at the University of Wisconsin-Madison Department of Bacteriology, On-line textbook of Bacteriology
Figure 1. Endotoxins can influence cell growth, cell differentiation, contractility and protein expression. They trigger the release of tumor necrosis factor (TNF), interleukins-1,6 and 8 and the production of platelet-activating factors, Prostaglandin E and Thromboxane A.1,2
Endotoxins Can Be Measured Using Two Methods
- 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 based on a Limulus amoebocyte lysate (LAL) and a synthetic color-producing substrate. The LAL assay is used for the routine check of LPS in biological solutions and was the first detection method to be certified by the FDA. LPS contamination is usually expressed in endotoxin units (EU). Typically, 1 mg LPS corresponds to 1–10 EU.
Other products of interest
In addition to DNA and RNA isolation products, Life Technologies offers an expansive portfolio of solutions for analysis and quantitation of tissue and cultured cell genomic DNA.