User manual

Section 3- Applications
• 260/230 - ratio of absorbance at 260 nm and 230 nm. This is a secondary measure of nucleic acid purity.
The 260/230 values for a “pure” nucleic acid are often higher than the respective 260/280 values and 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.
• Baseline correction - if selected, the default wavelength for the bichromatic normalization is 340 nm. The
user can manually enter a different wavelength for the bichromatic normalization of the absorbance data. In
either case, the baseline is automatically set to the absorbance value of the sample at the selected
wavelength. All wavelength data will be referenced off this value.
Note: If a baseline correction is not selected, the spectra may be offset from the baseline and the calculated
concentration will change accordingly.
Making Nucleic Acid Measurements
1. Select the Nucleic Acid application from the main menu. If the wavelength verification window appears,
ensure the arm is down and click OK.
2. Select the type of sample to be measured from the Type drop-down list. The default setting is DNA-50.
3. Choose the concentration units from the drop-down list adjacent to the color coded concentration box. The
default units are ng/µL.
4. A default wavelength of 340 nm is automatically used for a bichromatic normalization. Select an alternative
reference wavelength or choose not to have the spectrum normalized by de-selecting the baseline
correction box.
- Select the file drop-down option Use current settings as default as a convenient way to limit set-up time
for each new workbook.
5. Select Add to report to automatically include all measurements in the current report. The default setting is
for all samples to be added to reports. The Add to report checkbox must be selected prior to a
measurement to save the sample data to a workbook.
6. Select Overlay spectra to display multiple spectra at a time.
7. Establish a blank using the appropriate buffer. The blank solution generally is the buffer that the molecule of
interest is suspended or dissolved in. This solution should be the same pH and of a similar ionic strength as
the sample solution.
- Pedestal Option: Pipette 1-2 µL of the appropriate blanking solution onto the bottom pedestal, lower the
arm and click the Blank button.
- Cuvette Option (Model 2000c only): Insert the cuvette noting the direction of the light path indicated by
the etched arrow. The optical beam (2 mm) is directed 8.5 mm above the bottom of the cuvette. Refer to
the cuvette manufacturer for volume recommendations.
Note: The arm must be down for all measurements, including those made with cuvettes. It is
recommended that cuvettes be removed from the instrument prior to making a pedestal
measurement to ensure that the pedestal arm can move to the proper starting position.
8. Enter a Sample ID in the appropriate field, load the first sample as described for the blank above and click
Measure.
Note: A fresh aliquot of sample should be used for each measurement.
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