How to evaluate the quality of plasmid DNA is reliable?

There are many steps in the plasmid extraction process that affect the final yield and quality. How do you assess the yield and quality of plasmid DNA?

The current use of spectrophotometric methods for quantification of plasmid DNA and analysis of plasmid DNA yield and quality by agarose gel electrophoresis are the two most common methods.

The concentration of nucleic acid in the solution can be conveniently calculated by absorbance at 260 nm. The measurement results have good repeatability when the value of A260 is between 0.1 and 1.0, but when the value of A260 is less than 0.1 or greater than 1.0, the repeatability of the result will be significantly reduced. Moreover, readings above 3.0 are unusable and may potentially lead to underestimation of DNA quality. Therefore, in order to obtain reliable DNA spectrophotometric quantitative results, the reading of A260 needs to be between 0.1 and 1.0. Quantitative analysis using agarose gel electrophoresis may be more reliable when detecting small amounts of DNA.

There are many possible factors that cause lower yields and quality. To identify the problem, a small portion of the sample was retained for each purification step and analyzed by agarose gel electrophoresis.

As shown in each experimental protocol and in the following table, eluted lysate (sample 1), effluent (sample 2), mixed components of QC wash buffer (sample 3), and QF/QN buffer eluted product (sample) 4) Take out some samples in each. The nucleic acid was precipitated using 1 volume of isopropanol and the precipitate was washed with 70% ethanol, fully evaporated to dryness and resuspended in 10 μl of pH 8.0 TE buffer.

Table 1 Sample volume required for agarose gel analysis

In a 1% agarose gel*, 2 μl of each sample in each plasmid purification step was added for electrophoresis.

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