Measurement of Micro-Volumes of Proteins and Nucleic Acid

Download PDF October 4, 2017

Introduction

Micro-Volumes of Proteins and Nucleic Acid
V-630BIO UV-Visible Spectrophotometer

The SAH-769 One Drop accessory is used to measure micro-volumes of proteins and nucleic acids, either 0.6μL or 5 μL of sample with a 1 mm or 0.2 mm optical pathlength, respectively. The precise optical pathlength is controlled by covering the sample with a glass cover integrated into the cell unit. The cell and glass cover can be cleaned, simply by wiping with a laboratory tissue. This simple system can be used to measure samples quickly and easily. The shorter optical pathlength allows for measurements of high concentration samples without further dilution.

The V-630 Bio is double-beam spectrophotometer for high precision and accurate sample measurements, and can be operated using the cross-platform Spectra Manager or intelligent Remote Module (iRM) with color touch sensitive display. In either case, the Bio software includes standard programs for life science analyses. The Protein/Nucleic Acid program calculates either the protein or nucleic acid sample concentration from the ratio of 260/280nm absorbance. The Temperature Control program with optional Peltier thermostatted cell holder can be used for DNA thermal melting experiments.

This application note demonstrates the use of the one drop cell accessory with the V-630BIO system to measure small volume DNA samples.

Experimental

Measurement Conditions
Scan Speed200 nm/minData Interval0.5 nm
Bandwidth1.5 nmResponseMedium

Solutions of calf thymus DNA in KH2PO4/NaOH buffer (pH 7) at several concentrations were measured by using cells with 1 and 0.2 mm pathlengths. The measurement procedure for the SAH-760 One Drop accessory is shown in Figure 1.

Figure 1. Measurement procedure using the SAH-769 One Drop Cell.

Keywords

UV-0018, V-630BIO, UV-Visible/NIR, Life Science, Biochemistry, SAH-769 One Drop, Microsampling, nucleic acid, DNA, RNA, Protein

Results

The absorption spectra of various concentrations of calf thymus DNA using 1 and 0.2 mm pathlengths are shown in Figures 2 and 3, respectively.

Figure 2. Absorption spectra of calf thymus DNA
using a 1 mm pathlength cell.
Figure 3. Absorption spectra of calf thymus DNA using a 0.2 mm pathlength cell.

The corresponding calibration curves using the absorbance maxima at 260 nm are shown in Figures 4 and 5. Both curves demonstrate good linearity.

Figure 4. DNA absorbance calibration curve in a 1 mm pathlength cell.
Figure 5. DNA absorbance calibration curve in a 0.2 mm pathlength cell.

The disc cell and cover glass were wiped clean after measuring the absorbance of the highest concentrated sample in the 1 mm pathlength (5 μL volume) cell. A 5μL solvent was then measured to evaluation sample cross-contamination of the disc cell. The DNA sample absorbance was 1.8832 while the solvent absorbance was 0.0002, indicating that wiping is enough to wash the sample from the cell.

About the Author

Leah Pandiscia received her PhD from Drexel University where she studied Biophysical Chemistry. She is a Spectroscopy Applications Scientist at JASCO.