Application Information Spectrophotometric quantification of nucleic acids using the nanovette microliter cell accessory with a DU 730 spectrophotometer Amy Yoder, Mary Blair, Kenny Abell Beckman Coulter, Inc., Indianapolis, IN, USA Introduction Nucleic acid quantification is one of the most important applications in molecular biology labs. The concentration and purity of double stranded DNA (dsdna), single stranded DNA (ssdna) and RNA are routinely determined utilizing UV/Vis spectroscopy. In UV/Vis spectroscopy, standard cuvettes with 1 cm pathlengths requiring 50 μl to 2000 μl volumes are typically used. In order to remain in the linear range of the spectrophotometer, dilution of the sample is often required which subsequently eliminates its use in downstream applications. The nanovette microliter cell addresses the shortcomings of traditional cuvettes by allowing measurement samples as small as 0.7 μl with concentrations as high as 5,000 ng/μl. This application note presents data on the outstanding linearity, dynamic range, reproducibility, and correlation to a standard cuvette of the nanovette microliter cell when used with Beckman Coulter s DU 730 spectrophotometer. Operation By reducing the measurement pathlength, the nanovette allows sample size to decrease from the typical milliliter to less than a microliter. Pathlength adjustment takes place via interchangeable lids that easily fit onto the top of the microliter cell. A 1 mm pathlength lid provides for a virtual dilution of 1:10; a 0.2 mm pathlength lid provides for a virtual dilution of 1:50. Because a shorter pathlength reduces the measured absorbance of the solution, it is critical that the spectrophotometer offers a wide dynamic range in order to make measurements over a wide range of concentrations. Used with Beckman Coulter s DU 730 spectrophotometer, the nanovette can measure dsdna concentrations ranging from 10 ng/μl to 5,000 ng/μl. Table 1 provides the measurement range of the nanovette when using the DU 730 spectrophotometer for different nucleic acid samples.* Table 1. nanovette Measurement Range with the DU 730 Sample 1 mm Lid 0.2 mm Lid Specific (Virtual Dilution Factor 10) (Virtual Dilution Factor 50) Total Detection Range Factor [ng/μl] [ng/μl] [ng/μl] dsdna 50 10 1,000 200 5,000 10 5,000 ssdna 37 7 740 150 3,700 7 3,700 ssrna 40 8 800 160 4,000 8 4,000 Oligo 30 6 600 120 3,000 6 3,000 * The ranges for ssdna, ssrna and Oligo are based on the results from dsdna. A-11074A
The size of a standard cuvette (12.5 mm x 12.5 mm x 53 mm), the nanovette fits easily into the cuvette holder of the DU 730. No alignment is required, although a small step must be attached to the bottom of the microliter cell to obtain the proper aperture center height (both the step and a small screwdriver are included with the nanovette). Once in position, the nanovette cell remains in place during filling, measuring and cleaning. This guarantees a continuously identical position of the aperture in the light beam and no variation in comparison to the reference measurement. Sample volume depends on the particular lid being used: the 0.2 mm pathlength lid accepts from 0.7 μl to 4.0 μl; the 1.0 mm lid accepts from 3.0 μl to 5.0 μl. Detailed operation instructions can be obtained from the nanovette user s guide, available at www.beckmancoulter.com/nanovette Experimental For all of the following tests, a stock solution was prepared by dissolving salmon testes DNA in nuclease free sterile water to a concentration of approximately 5,000 ng/μl. This solution was then serially diluted to provide multiple samples ranging from approximately 5,000 ng/μl to 10 ng/μl. For large templates, such as genomic DNA, it is necessary to heat the DNA before measurement to relax the structure in order to obtain a completely homogenous solution. The DNA used in this application was heated at 60 C for at least 15 minutes and then mixed thoroughly prior to measurements. Unless otherwise stated, the sample volume used in each measurement was 1.5 μl with the 0.2 mm pathlength lid and 4 μl with the 1.0 mm To determine the nucleic acid concentration in solution, the absorbance at wavelength 260 nm (A260) is used. The following function, derived from Lambert-Beer s law, is applied: Concentration = Sample Virtual (A260 A320) Specific Dilution Factor Factor In the case of dsdna, the sample specific factor is 50. The virtual dilution factor will be 50 or 10, depending on whether the 0.2 mm pathlength lid or the 1.0 mm pathlength lid, respectively, is used. In this formula, the background absorbance of 320 nm must be subtracted for accurate results. Rather than taking separate measurements, the 320 nm background correction feature was enabled on the DU 730 spectro photometer. This feature automatically measures and subtracts the absorbance at 320 nm from the obtained results. NOTE: Solution homogeneity is critical when measuring low-volume samples on a fiber-optic microcell such as the nanovette. Therefore, when using large templates such as genomic DNA, it is necessary to preheat the sample to 60 C for 15 minutes followed by thoroughly mixing to ensure the accuracy of the data obtained with the nanovette. Linearity To measure linearity, a stock solution was serially diluted to provide samples ranging from approximately 10 ng/μl to 5,000 ng/μl (see experimental). These samples were then measured using both the 1.0 mm pathlength lid and the 0.2 mm pathlength lid and the results plotted as figure 1 and figure 2, respectively. Note that each data point represents the average of 10 measurements. Linearity is excellent, evident by an R 2 of over 0.998 in both cases. Reproducibility Reproducibility of the nanovette was measured in two different ways. Method 1: Reproducibility using the same sample size 10 measurements each of the varied concentrations were made, with results included in table 2. The sample size for the 0.2 mm lid was 1.5 μl; the sample size for the 1.0 mm lid was 4 μl. 2
Figure 1 Figure 2 Table 2. Reproducibility results using same sample size Concentration (ng/μl) 9 48 96 201 537 1168 Coefficient of Variation 7.77% 1.31% 1.30% 0.83% 0.45% 1.11% Concentration (ng/μl) 226 584 1229 2498 2966 5192 Coefficient of Variation 2.16% 1.45% 0.49% 1.01% 0.58% 1.67% Method 2: Reproducibility with varied sample size The nanovette accepts sample sizes from 3 μl to 5 μl when using the 1.0 mm pathlength lid, and from 0.7 μl to 4 μl when using the 0.2 mm pathlength lid. In this experiment, 4 measurements of a single concentration were made at various volumes with each The reproducibility data shows excellent reproducibility across a wide concentration, with an expected decrease in reproducibility at the lower end of the measurement range (coinciding with nearing the noise floor of the spectrophotometer). Reproducibility with different volumes is also excellent, with a 0.82% difference from the mean using extreme sample volumes with the 1.0 mm pathlength lid and a 3.41% difference from the mean using the extreme sample volumes with the 0.2 mm Table 3. Reproducibility results with varied sample size Avg. Conc. % diff. Volume (ng/μl) CV from mean 5 μl 952 0.14% 0.31% 4 μl 948 0.42% 0.11% 3 μl 944 0.58% 0.51% Avg. Conc. % diff. Volume (ng/μl) CV from mean 3 μl 989 0.15% 1.78% 2 μl 1,009 0.12% 0.26% 1 μl 1,006 0.12% 0.11% 0.7 μl 1,023 0.12% 1.63% 3
Carryover Effects One of the primary benefits of the nanovette is improved throughput; the ability to quickly and easily clean the device is paramount. When cleaning the nanovette, the cell remains inside the spectro - photometer and the measurement window cleaned with a laboratory wipe. For the pathlength lid, a lintfree swab can be used to fully evacuate the area. However, due to the shape of the lid, compressed air is recommended over the lint-free swab. In this application note, the cell and the lid were wiped with a lint free swab followed by drying with compressed air between each sample. Carryover effect was assessed by measuring 3 sets of 3 samples of DNA followed by 3 samples of buffer solution. Results are shown in figure 3 for the 1.0 mm pathlength lid and figure 4 for the 0.2 mm Correlation to a Standard Cuvette To test the correlation of the nanovette with a standard quartz cuvette (pathlength = 10 mm), a dilution series of dsdna was measured over a broad range. 10 measurements with the nanovette were taken at each concentration against 3 measurements with a standard cuvette, with the results plotted as figure 5 for the 1.0 mm pathlength lid and figure 6 for the 0.2 mm Note that in order to compare the data, the dsdna measured in the standard quartz cuvette had to be diluted accordingly to fall within the absorbance range of the instrument. 2.50 1,000 ng/μl 2.50 5,000 ng/μl Absorbance 260 mm 2.00 1.50 1.00 0.50 Absorbance 260 mm 2.00 1.50 1.00 0.50 0.00 0.00 sample 1 sample 2 sample 3 water 1 water 2 water 3 sample 4 sample 5 sample 6 water 4 water 5 water 6 sample 7 sample 8 sample 9 water 7 water 8 water 9 sample 1 sample 2 sample 3 water 1 water 2 water 3 sample 4 sample 5 sample 6 water 4 water 5 water 6 sample 7 sample 8 sample 9 water 7 water 8 water 9 Figure 3 Figure 4 The data shows the importance of proper cleaning of the nanovette. Empirical results have shown that proper cleaning of the nanovette will result in virtually no carryover. 1200 6000 1000 5000 Concentration (ng/μl) 800 600 400 Standard Cuvette 1.0 mm Lid Concentration (ng/μl) 4000 3000 2000 Standard Cuvette 0.2 mm Lid 200 1000 0 0 Figure 5 Figure 6 4
As expected, the longer 1.0 mm pathlength lid does provide greater accuracy, but both lids provide excellent results over their respective ranges. Conclusion This application note demonstrates the performance of the nanovette microliter cell accessory when used with the DU 730 spectrophotometer. Excellent linearity and reproducibility across a wide concentration range, reproducibility within a wide variety of sample sizes, minimal carryover effect, and correlation to a standard cuvette provides confidence regarding any potential DNA or protein application using the nanovette with the DU 730 spectro - photometer. The nanovette is a useful accessory to the biology laboratory, providing fast, easy, and accurate measurements. Australia, Gladesville (61) 2 9844-6000 Canada, Mississauga (1) 905 819 1234 China, Beijing (86) 10 6515 6028 Czech Republic, Prague (420) 267 00 85 13 Eastern Europe, Middle East, North Africa, South West Asia: Switzerland, Nyon (41) 22 365 3707 France, Villepinte (33) 1 49 90 90 00 Germany, Krefeld (49) 2151 33 35 Hong Kong (852) 2814 7431 India, Mumbai (91) 22 3080 5101 Italy, Cassina de Pecchi, Milan (39) 02 953921 Japan, Tokyo (81) 3 5530 8500 Korea, Seoul (82) 2 404 2146 Latin America (1) (305) 380 4709 Mexico, Mexico City (001) 52 55 9183 2800 Netherlands, Mijdrecht (31) 297 230630 Puerto Rico (787) 747 3335 Singapore (65) 6339 3633 South Africa/Sub-Saharan Africa, Johannesburg (27) 11 805 2014/5 Spain, Madrid (34) 91 3836080 Sweden, Bromma (46) 8 564 85 900 Switzerland, Nyon (41) 0800 850 810 Taiwan, Taipei (886) 2 2378 3456 Turkey, Istanbul (90) 216 309 1900 UK, High Wycombe (44) 01494 441181 USA, Fullerton, CA (1) 800 742 2345 B2007-7786 www.beckmancoulter.com 2007 Beckman Coulter, Inc. BMR-PRINTED IN U.S.A.