Abstract Concentrations of DNA in solution (31µg/mL and 144µg/mL) were measured with a spectrometer and UV/VIS light source in a SPT-2. Due to the 1cm pathlength, use of a SPT-2 does not require a pre-measurement dilution within this concentration range, thus a potential source of error was eliminated. Due to the design of the SPT-2, routine measurements can be taken with samples of 10µL in standard 200µL PCR tubes. If caution is taken with regard to the location of the probe tip within a PCR via, measurements remain repeatable in sample sizes as small as 5µL.
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Experimental Procedure
Standard solutions of DNA (Sigma D1626) were prepared gravimetrically
using 18.2MΩ/cm ultrapurified water as a solvent. Solutions were
prepared between 0.0µg/mL and 143.9µg/mL.
Measurements were taken in triplicate using an SPT-2. The SPT-2 was connected to a Tidas I spectrometer (WPI #TIDAS-1) and a UV/VIS light source (WPI #D4H).
Data were collected in 1nm increments across the full range of the
instrument (190nm-720nm). The instrument was configured such that
reference measurements yielded an 80% total intensity. All measurements
utilized 18.2MΩ/cm ultrapurified water as a reference solution.
Results
Experimental results are presented in Table 1:
DNA [µg/mL]
|
Absorbance @260nm [AU]
|
0.00
|
-0.0061 ± 0.0001 |
| 30.83 |
0.5745 ± 0.0011 |
| 56.59 |
1.0309 ± 0.0020 |
| 85.49 |
1.4497 ± 0.0016 |
| 115.66 |
1.7170± 0.0025 |
| 143.94 |
1.8280 ± 0.0006 |
Table 1: DNA Concentrations and Resultant Absorbance Values
Since absorbance with respect to concentration follows the Beer-Lambert Law,
A= εlc
expected absorbance values were calculated from the DNA solution
concentrations. Literature values of ε for dsDNA are listed as
0.020µg/ml*cm.
Experimental data can be found in Figure 1 with the calculated
absorbance measurements indicated by a solid line. Absorbance
measurements are expressed at a 260nm wavelength. Deviation from the
theoretical value at higher absorbance values is a result of stray light
interference within the spectrometer.
Figure 1: Measured versus Theoretical Absorbance
Figure 2: Typical DNA Measurement (56.6µg/mL)
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