GenScript's plasmid DNA preparation services offer the most stringent quality control standards on the market, and services can be customized to your quality and quantity needs. Preps are offered at up to ≥ 90% supercoil and ≤ 0.01 EU/µg endotoxin levels. Here are the QC items we monitor when making plasmid preps:
|QC items||Specification||Analytical Method|
|Appearance||Clear, no visible particles||Visual inspection|
|A260/280||1.8-2.0||Nanodrop UV absorption|
|Concentration||On request, default 1 mg/ml||UV absorption|
|Supercoil content||For information only||Densitometry upon agarose gel electrophoresis|
|Residue RNA||Not visible||Visual inspection upon electrophoresis|
|Genomic DNA||Not visible||Visual inspection upon electrophoresis|
|Restriction Analysis||Conforms to reference||Enzyme digestion and electrophoresis|
|Endotoxin assay||Based on grade||LAL assay|
|Bioburden assay||No growth on agar plate after 48 hours||Bioburden|
Endotoxins deciphered: What role do they play in the plasmid prep process?
Q: What are endotoxins?
A: Endotoxins are found in the outer membrane of bacteria (gram-negative), such as E. coli. They are also known as lipopolysaccharides or lipoglycans. They consist of 2 separate parts, a lipid and a polysaccharide, that are joined by a covalent bond. The polysaccharide itself consists of 3 parts: The O-antigen, outer core, and inner core. Endotoxins themselves in bacteria contribute to the structural stability of the bacteria, both anchoring the integrity of the membrane and protecting the bacteria itself from certain stresses.
Q: Why should I care about endotoxins in my plasmid preps?
A: Endotoxins can have adverse effects on certain downstream applications following plasmid purification. If found endotoxins are found at high enough levels, they can influence the process of transfection of DNA into cells, leading to reduced efficiencies.
Endotoxins can also have adverse effects on gene therapy applications, as they can lead to several side effects such as fever in both animals and humans and can lead to misinterpretation of research results.
Q: What different options are offered by GenScript in terms of endotoxin levels?
A: Here at GenScript, we offer several different grades of endotoxin levels in our plasmid prep services to fit your needs. Our entry level plasmid prep, in terms of endotoxin QC, is our Research Grade plasmid prep service. This service does not include our endotoxin-level free preparation process, but includes several of our other stringent quality control measures. Our highest level of endotoxin QC is our Industrial Grade plasmid prep service. This service guarantees endotoxin levels that are ≤ 0.01 EU/µg, representing the highest stringency endotoxin QC in the market today. The QC for our HT plasmid prep service guarantees an endotoxin-free preparation process, but does not include an endotoxin assay (LAL test) to measure actual endotoxin levels. However, routine testing of the process finds that endotoxin levels are consistently < 0.1 EU/µg which is suitable for mammalian cell transfection.
Q: What does "Endotoxin-Free Process" and "Endotoxin Removal Process" mean?
A: Endotoxin-free refers to the materials and reagents being free of endotoxins. Endotoxin Removal Process refers to a separate step in the plasmid prep process that occurs at the end to remove as much endotoxin as possible using the addition of a chemical reagent.
Q: What different options are offered by GenScript in terms of endotoxin assay?
LAL Assay Quantitative LAL Assay Description
A gel clot assay used to determine whether test samples contain specific amounts of endotoxin. The sample and the limulus amebocyte lysate (LAL) reagent solution are mixed in an endotoxin-free tube and incubated at 37℃ for 60 minutes (Figure 1). After incubation, the tube is tilted to see whether the mixture clots. If the sample contains less endotoxin than the standard, the solution remains liquid; whereas if the endotoxin amount is higher than the standard, the mixture will clot at the bottom of the tube (Figure 2).
The endpoint chromogenic LAL test is a quantitative test for detection of gram negative bacterial endotoxins. A sample is mixed with the LAL supplied in the test kit and incubated at 37℃±1℃ for 10 minutes. A substrate solution is then mixed with the LAL-sample and incubated at 37℃±1℃ for an additional 6 minutes. The reaction is stopped with stop reagent. If endotoxin is present in the sample, a yellow color will develop. The absorbance of the sample can be determined spectrophotometrically at 405–410 nm. Since this absorbance is in direct proportion to the amount of endotoxin present, the concentration of endotoxin can be calculated from a standard curve.
Process & Results
Q: Should I worry about endotoxins for my research purposes?
A: Here is a table below showing the different applications for both our Research Grade Plasmid Prep (no endotoxin QC) and Industrial Grade Plasmid Prep (endotoxin levels ≤ 0.01 EU/µg3):