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Plasmid extraction is crucial for researchers. As gene vectors, plasmids are vital in vaccine research and gene therapy, where quality matters. Key quality factors include supercoiled proportion and endotoxin content. Regulations vary: FDA ≥80%, SMPA ≥85%, NMPA ≥90%, with <5% linear/open-circular DNA. Clients often prefer ≥95% supercoiled. Endotoxin levels are: research (>50 EU/µg), transfection (<50 EU/µg), and endotoxin-free (<0.1 EU/µg).

Supercoiled Plasmid DNA

Plasmids from Escherichia coli include supercoiled (SC), open-circular (OC), and linear (L) DNA. High SC proportion boosts transfection efficiency and expression. For example, an 85.45% SC plasmid's transfection is 2.5x higher, and a 93.28% SC plasmid's HGF expression is 1.8x higher, confirming SC DNA's superiority.

Open-circular DNA: When one strand of double-stranded DNA breaks, the supercoiled structure becomes loose, forming an open-circular plasmid with a larger volume that migrates the slowest during electrophoresis and is positioned at the top.

Linear DNA: When both strands of plasmid DNA break, a linear structure forms, with a relatively smaller volume that migrates faster than open-circular DNA but slower than supercoiled DNA, positioned second.

Supercoiled DNA: Two helixes intertwine, twisting into a supercoiled structure based on the DNA helix. It has the smallest volume and migrates the fastest during electrophoresis, positioned at the bottom.

Quantitative Analysis of Supercoiled Proportion

Labs use agarose gel electrophoresis (AGE) and Gel-Pro Analyzer to judge supercoiled proportion via electrophoresis images. One image shows 97.539% SC DNA, while another shows 63.833% SC and 36.167% OC DNA (linear DNA not visible).

The Secret of Endotoxin in Plasmids

Endotoxin is a component of the outer layer of the cell wall of Gram-negative bacteria, released after bacterial death or lysis, containing toxic lipopolysaccharide A. During plasmid extraction, endotoxin enters the lysis solution, affecting transfection efficiency and triggering nonspecific immune responses, leading to false positives in experiments.

Quantitative and Qualitative Analysis of Endotoxin

The Limulus amebocyte lysate (LAL) test detects endotoxin via agglutination with 2-keto-3-deoxyoctonic acid (KDO) in the lipopolysaccharide (LPS). It's qualitative and semi-quantitative, indicating endotoxin presence through coagulation or color change when mixed with samples.

Yaohai Bio-Pharma offers endotoxin-free supercoiled plasmids. Outsourcing to Yaohai boosts transfection and expression efficiency. Delivered plasmids have a ≥95% SC proportion (avg. 85%±10%) and endotoxin levels ≤0.1, ≤0.01, or ≤0.005 EU/μg.

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