Quality Identification Song after Nucleic Acid Extraction

Quality Identification Song after Nucleic Acid Extraction

Nucleic acid purification has been widely used in molecular biology laboratories. How to obtain high quality and high purity DNA or RNA samples is crucial to the smooth performance of downstream experiments. Therefore, the identification of purified nucleic acids is also an essential step.

Purity evaluation

Spectrophotometry

Principle: Since the bases in nucleic acids all have conjugated double bonds, they have UV absorption properties. Nucleic acids have a typical absorption curve in the UV spectral region, with their absorption peak at 260 nm and absorption trough at 230 nm. The absorption peak of protein in the UV region is at 280 nm, so the UV absorption spectral data of nucleic acid is one of the important basis for nucleic acid identification.

Choose the correct dilution: make sure the OD260 value is between 0.1 and 1.0.

DNA Yield=OD260*dilution factor*50ng/μl, RNA Yield=OD260*dilution multiple*40ng/μl

DNA purity

OD260/OD280 = 1.7-1.9, OD260/OD230 = about 2.0, indicating good purity.

DNA purity issues.

OD260/OD280 <1.7, indicating protein contamination.

OD260/OD280>1.9, indicates partial degradation or RNA contamination.

OD260/OD230 <2.0, indicates salt ions, organic macromolecule residues.

RNA purity

OD260/OD280=1.9-2.1, OD260/OD230= about 2.0, indicating good purity.

RNA purity issues.

OD260/OD280 <1.9, indicating protein or genomic residues.

OD260/OD280>2.1, indicating partial degradation of RNA.

OD260/OD230 < 2.0, indicating salt ion, macromolecule residue.

Concentration Detection

There are various detection methods for nucleic acid concentration detection, this article mainly introduces the fluorescence photometric method.

Principle: Fluorescence photometry uses nucleic acid dyes embedded in the base plane to make nucleic acid which is not fluorescent in itself fluoresce orange under UV excitation, and the fluorescence intensity is proportional to the nucleic acid content. Finally, the standard curve is used as the chord to play the music by the fluorescence intensity.

Nucleic Acid Integrity Assessment

Agarose gel electrophoresis is the traditional method used to assess the integrity of nucleic acid products after extraction. For genomic DNA, clear single integrity bands are obtained; for high quality total RNA, clear 28s, 18s and 5s bands are obtained after electrophoresis.

Genetic DNA Electrophoretogram

Total RNA Electrophoretogram

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