Small Volume DNA Thermal Melting using a One Drop Accessory with Capillary Cell

Download PDF January 25, 2018

Introduction

Small Volume DNA Thermal Melting Analysis
V-630 UV-Visible Spectrophotometer

Thermal melting studies are commonly carried out in biochemical and biotechnology fields in order to obtain information regarding a biological sample’s thermodynamic properties. These properties can further elucidate the sample’s thermal stability and possible changes in structure and/or binding.

For many biological samples, it is much more convenient, cost effective, and efficient to use micro-volumes for structural and quantitative studies. Small volume DNA thermal melting analysis was made using the One Drop capillary jacket with a disposable quartz capillary cell, optical pathlength  0.5 mm and sample volume 3 µL.

This application note demonstrates the use of the one drop capillary jacket accessory for biological sample absorption measurements.

Experimental

Measurement Conditions
Measurement Wavelength260 nmData Acquisition Interval0.1°C
Temperature Gradient1°C/minResponseFast
Start Setting3 seconds +/-0.10°C

The measurement procedure is illustrated in Figure 1. The sample is drawn into the cell via capillary action and both edges of the capillary cell are sealed which helps to prevent volatilization of the sample. The capillary cell is then inserted into the capillary jacket and placed into a Peltier thermostatted cell holder. The jacket has a temperature sensor insertion port to ensure accurate measurements of the actual sample temperature.

Figure 1. Measurement procedure for filling the capillary cell.

Keywords

UV-0008, V-630, UV-Visible/NIR, Microsampling, ETCS-761 Water-cooled Peltier thermostatted cell holder with stirrer, One Drop, Capillary jacket, Biochemistry, DNA thermal melting, Temperature ramping

Results

Figure 2 shows the result of measurement of Poly (dA-dT)-Poly(dA-dT). The absorption measurements were obtained in both the 10 mm rectangular cell (green) and the microsampling capillary cell (blue). The calculated melting temperature of Poly (dA-dT)-Poly(dA-dT) was 63.8ºC and 63.9ºC for the rectangular and capillary cells, respectively.

Figure 2. Thermal melting absorption data in a 10 mm rectangular cell (green) and capillary jacketed cell (blue).

Conclusion

The results illustrate that the One Drop capillary cell and jacket are as reliable as the 10 mm rectangular cell for obtaining absorption measurements on extremely small volumes.

About the Author

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