Micro-volume Analysis of a λ DNA Using a One Drop Accessory

Download PDF September 12, 2017

Introduction

FP-8200 Spectrofluorometer

For many biological samples, it is much more convenient, cost effective, and efficient to use microvolumes for structural and quantitative studies.  The SAF-850 One Drop is a microsampling accessory that enables fluorescence measurements to be obtained with only 5 µL of sample. The highly reproducible baseline allows for either simple spectrum measurements or quantitative analysis of multiple samples.

This application note demonstrates the use of the One Drop accessory to obtain fluorescence data and create a calibration curve of λDNA labeled with PicoGreen®.

Experimental

Measurement Conditions

Fluorescence SpectraCalibration
Excitation Wavelength485 nmExcitation Wavelength480 nm
Excitation Bandwidth10 nmEmission Bandwidth523 nm
Emission Bandwidth10 nmExcitation Bandwidth20 nm
Data Interval0.5 nmEmission Bandwidth20 nm
Response Time2 secResponse Time1 sec
Scan Speed100 nm/minSensitivity620 V
Sensitivity700 V
Figure 1. Flow chart illustrating the sample preparation procedure using the One Drop accessory.

Keywords

FP0010, FP-8200, Fluorescence, Microsampling, SAF-850 One Drop, FP0010

Results

The emission spectra of λDNA for a variety of concentrations is shown in Figure 2 and indicates that the maximum emission wavelength is 523 nm.

Figure 2. Emission spectra of λDNA labeled with PicoGreen®.

The fluorescence intensity at the maximum emission wavelength (523 nm) was then measured five times for each sample to ensure measurement reproducibility. Table 1 shows these measurement reproducibility results for each sample concentration.

Table 1. Measurement reproducibility results.

Concentration [ng/mL]12345AverageSDCV(%)
053.3 52.4 55.9 53.1 55.2 54.0 1.49 2.8
163.6 68.1 65.9 66.5 65.1 65.8 1.68 2.6
5110.3 106.7 105.1 104.0 110.0 107.2 2.86 2.7
10157.6 155.5 156.1 153.0 151.7 154.8 2.39 1.5
50447.1 465.3 460.2 455.8 469.0 459.5 8.56 1.9
100865.9 856.9 848.3 850.6 853.9 855.1 6.86 0.8
5003842.7 3831.0 3858.0 3828.9 3811.0 3834.3 17.42 0.5
10007766.2 7992.1 7925.3 7972.8 7805.7 7892.4 101.15 1.3

A calibration curve was then created using the average value for each concentration from the reproducibility results in Table 1 and is shown in Figure 3. The equation of the line fit to the calibration curve is y = 7.780 • c + 57.5211. The correlation coefficient is 0.9999 and the standard error is 6.819, indicating a good fit.

Figure 3. Calibration curve of λDNA labeled with PicoGreen®.

Conclusion

This application note demonstrates the measurement reproducibility of the One Drop microsampling accessory by obtaining a calibration curve with good linearity over a wide concentration range.

Required Products and Software

  • FP-8200/8300/8500/8600/8700 Spectrofluorometer
  • SAF-850/851 One-Drop Measurement Accessory

About the Author

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