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How to determine plasmid DNA concentration and purity?
We use horizontal agarose gel electrophoresis, adding 0.5 µg/mL EB to the gel, and include a standard sample of known concentration. After electrophoresis, compare the brightness under UV light to judge concentration and purity. This method more directly and accurately assesses whether the sample contains genomic DNA, RNA, etc., and can also distinguish different conformations of the extracted plasmid DNA.
The three conformations of plasmid DNA refer to: during the plasmid extraction process, due to various factors, the supercoiled covalently closed circular (CCC) plasmid can have one strand break, becoming open circular (OC). If both strands break, it becomes linear (L). These three forms have different migration rates. Typically, supercoiled (SC/CCC) migrates fastest, followed by linear (L), and open circular (OC) migrates slowest.
Using a UV spectrophotometer or the EB-standard DNA comparison method only detects the concentration of the extracted product. However, due to potential interference from RNA, protein, genomic DNA, etc., in the extracted plasmid DNA, the concentration value obtained may be meaningless.
What are the requirements for plasmid DNA used for sequencing?
General requirements for sequencing plasmid DNA:
(1) High DNA purity (A260/A280 ratio 1.6–2.0). It must not contain mixed templates, RNA, genomic DNA, or proteins.
(2) Dissolved in ddH₂O. The solution must not contain impurities such as salts or chelators like EDTA, as these will interfere with the sequencing reaction.