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Home / Applications / Quantitative Analysis of a λ DNA Using a One Drop Accessory

  • Industry

  • Technique

Quantitative Analysis of a λ DNA Using a One Drop Accessory

By Heather Haffner

PDF IconDownload This Application

January 5, 2024

Introduction

FP-8200 Spectrofluorometer
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
Flow chart illustrating the sample preparation procedure using the One Drop accessory
Figure 1. Flow chart illustrating the sample preparation procedure using the One Drop accessory.

Keywords

260-FP-0010, 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.

Emission spectra of λDNA labeled with PicoGreen®
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.

Calibration curve of λDNA labeled with PicoGreen®
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
This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.

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About the Author

JASCO Application Note

Quantitative Analysis of a λ DNA Using a One Drop Accessory

Introduction

FP-8200 Spectrofluorometer
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
Flow chart illustrating the sample preparation procedure using the One Drop accessory
Figure 1. Flow chart illustrating the sample preparation procedure using the One Drop accessory.

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.

Emission spectra of λDNA labeled with PicoGreen®
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.

Calibration curve of λDNA labeled with PicoGreen®
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.

Keywords

260-FP-0010, FP-8200, Fluorescence, Microsampling, SAF-850 One Drop, FP0010

Required Products and Software

  • FP-8200/8300/8500/8600/8700 Spectrofluorometer
  • SAF-850/851 One-Drop Measurement Accessory
This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.
28600 Mary’s Court, Easton, MD 21601 USA • (800) 333-5272 • Fax: (410) 822-7526 • jascoinc.com/applications

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