Question 12 of 20 Fundamentals Interpretation
What do the A260 to A280 and A260 to A230 ratios tell you about a nucleic acid prep?
Short answer
Nucleic acids absorb most at 260 nm and proteins at 280 nm, so the 260 to 280 ratio flags protein carryover. A ratio near 1.8 is accepted as pure DNA and near 2.0 as pure RNA. The 260 to 230 ratio, normally 2.0 to 2.2, falls when organic solvents, guanidine or other extraction reagents remain.
Why
These ratios are a quick purity screen, not a quality measure. Thermo Fisher's technical note explains that residual chemicals can both overestimate the measured concentration and spoil downstream reactions. A low 260 to 230 ratio often means carryover of guanidine salts, phenol or ethanol from the extraction, any of which can inhibit PCR or reverse transcription.
Looking at the whole spectrum helps. The trough should sit near 230 nm, and a contaminant shifts it. Phenol gives a distinctive peak near 270 nm.
What these numbers cannot tell you is whether the molecules are intact. A completely degraded RNA sample can have a perfect 260 to 280 ratio, because the bases still absorb. Integrity needs a different method, such as capillary electrophoresis giving an RNA integrity number.
Absorbance also cannot distinguish DNA from RNA from free nucleotides, so a dye-based assay such as a fluorescent double-stranded DNA kit gives a more accurate concentration for a mixed sample. In practice, use absorbance to spot contamination, a fluorescent assay for concentration and electrophoresis for integrity.
A strong answer covers
- 260 nm for nucleic acid, 280 nm for protein, 230 nm for solvents
- About 1.8 for DNA and 2.0 for RNA, with 260 to 230 of 2.0 to 2.2
- Low 260 to 230 suggests guanidine, phenol or ethanol carryover
- Ratios say nothing about integrity
- Fluorescent assays give better concentrations for mixed samples
Common mistakes
- Treating a good 260 to 280 ratio as evidence that RNA is intact
- Using absorbance concentration for a sample that contains both DNA and RNA
- Ignoring a low 260 to 230 ratio before a sensitive enzymatic reaction
The interviewer might ask next
Which measurement would you add before using this RNA for library preparation?