Section 5- Nucleic Acids
5. Nucleic
Acids
Nucleic acid samples can be readily checked for concentration and quality using the NanoDrop
®
ND-1000 Spectrophotometer. To
measure nucleic acid samples select the ‘Nucleic Acid’ application module.
Sample Volume Requirements
Field experience has indicated that 1ul samples are sufficient to ensure accurate and reproducible results when measuring aqueous
nucleic acid samples. However, if you are unsure about your sample or your pipettor accuracy, a 1.5-2ul sample is recommended to
ensure that the liquid sample column is formed and the light path is completely covered by sample.
Measurement Concentration Range
The NanoDrop
®
ND-1000 Spectrophotometer will accurately measure dsDNA samples up to 3700 ng/ul without dilution. To do this, the
instrument automatically detects the high concentration and utilizes the 0.2mm pathlength to calculate the absorbance.
Detection
Limit
(ng/ul)
Approx.
Upper Limit
(ng/ul)
Typical Reproducibility
(minimum 5 replicates)
(SD= ng/ul; CV= %)
2
3700 ng/ul (dsDNA)
3000 (RNA)
2400 (ssDNA)
sample range 2-100 ng/ul:
±
2 ng/ul
sample range >100 ng/ul:
±
2%
Unique Screen Features
Sample Type:
used to select the (color-keyed) type of nucleic acid being measured. The user can select ‘DNA-50’ for dsDNA, ‘RNA-
40’ for RNA
,
‘ssDNA-33’ for single-stranded DNA, or ‘Other’ for other nucleic acids. The default is DNA-50. If ‘Other’ is selected, the
user can select an analysis constant between15-150. When navigating amongst the three general sample types within the Nucleic
Acids module, the last constant value entered within the ‘Constant’ sample type will be retained. See the “Concentration Calculation
(Beer’s Law)” Appendix for more details on this calculation.
λ
and
Abs:
the user selected wavelength and corresponding absorbance. The wavelength can be selected by moving the cursor or
using the up/down arrows to the left of the wavelength box.
Note: The user-selected wavelength and absorbance are not utilized in any calculations.
A260:
absorbance of the sample at 260 nm represented as if measured with a conventional 10 mm path. Note: This is 10X the
absorbance actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0.2 mm path length.
A280:
sample absorbance at 280 nm represented as if measured with a conventional 10 mm path. Note: This is 10X the absorbance
actually measured using the 1 mm path length and 50X the absorbance actually measured using the 0.2 mm path length.
260/280:
ratio of sample absorbance at 260 and 280 nm. The ratio of absorbance at 260 and 280 nm is used to assess the purity of
DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the
ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or
near 280 nm. See “260/280 Ratio” section of the Troubleshooting section for more details on factors that can affect this ratio.
260/230:
ratio of sample absorbance at 260 and 230 nm. This is a secondary measure of nucleic acid purity. The 260/230 values for
“pure” nucleic acid are often higher than the respective 260/280 values. They are commonly in the range of 1.8-2.2. If the ratio is
appreciably lower, this may indicate the presence of co-purified contaminants.
ng/ul:
sample concentration in ng/ul based on absorbance at 260 nm and the selected analysis constant. See the “Concentration
Calculation (Beer’s Law)” in the appendix for more details on this calculation.
5-1
