HPLC-UV DNA Quantification Protocol

The concentration of DNA was quantified by HPLC-UV analysis of dG in the enzymatic hydrolysate of DNA. The amount of DNA was calculated from the dG content by assuming that 1 mg of DNA contained 3 μmol of nucleotides and that dG accounted for 22% of the total nucleotides in DNA [36 (link)]. The experiment was conducted on a Thermo Finnigan HPLC system with a diode array detector. Gradient elution was performed using a Luna C18 column (4.6×250 mm, 5 μm in particle size, Phenomenex, Torrance, CA) eluted with deionized water (solvent A) and methanol (solvent B) at a flow rate of 0.7 mL/min. The UV signal was monitored at 254 nm. The solvent composition began at 5% B followed by linear increase to 22% B over 15 min. Then the solvent B was raised to 80% over 2 min followed by an isocratic elution for 3 min. Subsequently, the percentage of the solvent B was brought back to 5% over 2 min, followed by an equilibration for 15 min.

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Li L., Li S., Sun G., Peng R., Zhao L, & Zhong R. (2015). Influence of the Expression Level of O6-Alkylguanine-DNA Alkyltransferase on the Formation of DNA Interstrand Crosslinks Induced by Chloroethylnitrosoureas in Cells: A Quantitation Using High-Performance Liquid Chromatography-Mass Spectrometry. PLoS ONE, 10(3), e0121225.

Publication 2015

Finnigan HPLC system Luna C18 column

Enzymatic Hplc Methanol Nucleotides Solvent

Corresponding Organization :

Other organizations : Beijing University of Technology

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Variable analysis

independent variables

Concentration of DNA

dependent variables

Amount of DNA

control variables

HPLC-UV analysis
Enzymatic hydrolysate of DNA
Luna C18 column (4.6×250 mm, 5 μm in particle size)
Gradient elution using deionized water (solvent A) and methanol (solvent B) at a flow rate of 0.7 mL/min
UV signal monitored at 254 nm
Solvent composition began at 5% B followed by linear increase to 22% B over 15 min, then raised to 80% over 2 min, and brought back to 5% over 2 min

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