NanoPhotometer
®
N120/NP80/N60/N50/C40 User Manual Version 4.3.1
69
Ratios
Reactions utilizing nucleic acids often require minimum purity standards. Common
contaminants of nucleic acid samples include: proteins, organic compounds, and other.
Based on the common contaminants of nucleic acid samples, the 260/280 and 260/230
ratios are calculated for nucleic acids to give an indication of the purity of the samples. Pure
DNA and RNA preparations have expected 260/280 ratios of 1.8 and 2.0 respectively.
An elevated absorbance at 230 nm can indicate the presence of impurities as well; 230 nm
is near the absorbance maximum of peptide bonds and also indicates buffer contamination
since TRIS, EDTA and other buffer salts absorb at 230nm. When measuring RNA samples,
the 260/230 ratio should be > 2.0; a ratio lower than this is generally indicative of
contamination with guanidinium thiocyanate, a reagent commonly used in RNA purification
and which absorbs over the 230-260 nm range. If a ratio is detected out of the acceptable
range an alert icon is shown in the results/table area. A push on the alert icon shows
additional information. The ranges for acceptable ratio values can be defined in preferences.
The ratios are calculated with or without background correction according to if the
background correction is activated during the measurements or not as follows:
Without background correction:
260/280 ratio =
A
260
A
280
260/230 ratio =
A
260
A
230
With background correction:
260/280 ratio =
A
260
- A
BKG
A
280
- A
BKG
260/230 ratio =
A
260
- A
BKG
A
230
- A
BKG
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