Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

1. Procedure for DNA Quantitation Turn ON the instrument. Open the BioSpec-nano software from the PC desktop. Click on the Simple Nucleic Acid -dsDNA tab (Figure 1). The software will establish communication with the instrument and run an initialization sequence. This verifies that the instrument is performing according to specifications. Select 0.7 or 0.2 mm on the pathlength slider. This selects the pathlength and all calculations are based on the pathlength selected. Next in BioSpec-nano software click Setup-Autowiping setup. Select 1 or 2. Pipette 2 μL (for 0.7 mm) H2O or buffer on the pedestal. Click blank or F6 for blank measurement. Every time a new pathlength is selected it is necessary to record a blank. Pipette 2 μL of sample on the pedestal. Click Start in the software or push the start button on the instrument. The Upper window moves to make contact with the sample droplet. Xenon flashes can be observed. Repeat the same sample 3 times to determine reproducibility. All data recorded is saved as .bud file. To save as a .pdf or .csv file click save pdf/csv or F9. Results can also be saved as a .csv file by using the appropriate selection in the save tab. 2. Protocol for Protein Quantitation Select Protein Quantitation tab (Figure 2). Enter the extinction coefficient e280, if known. If e280 is not known calculate it using e280Calc. Select 0.7 or 0.2 mm on the pathlength slider. Click Setup-Autowiping setup. Select 2 or above. Pipette 3 μL (for 0.2 mm) H2O or buffer on the pedestal. Click blank or F6 for blank measurement. Pipette 3 μL of protein sample on the pedestal. Click Start in the software or push the start button on the instrument. Repeat the same sample 3 times to determine reproducibility. All data recorded is saved as .bud file To save as a .pdf or .csv file click save pdf/csv or F9. 3. Representative Results: 1. Results for DNA Quantitation Figure 3 shows the data from measurements on a typical double stranded DNA sample. The concentration and OD 260/280 ratio calculated are also shown. Figure 4 contains reproducibility test results for DNA quantitation. The data in Figures 3 and 4 shows results are highly reproducible as evident from the low relative standard deviation of 0.26%. Note 1: Inaccuracy in DNA quantitation can arise from the presence of buffer components which may absorb in the UV region. It is also necessary to use a homogenous sample solution. As the amount of sample being pipetted is only 1-2 μL, careful pipetting of accurate volumes without introduction of any bubbles is required. 2. Results for Protein Quantitation Figure 5 represents protein concentration analysis for BSA. The μg/ mL protein concentration is based on the e280 value. Figure 6 shows reproducibility analysis. It is evident from the relative standard deviation of 0.51% that the BioSpec-nano measures concentration precisely – not only for DNA but also for proteins. Note 2: Factors affecting protein concentration Accurate concentration determination of protein is feasible when there are minimum effects from instability caused by pH, buffers, temperature, or additives. Most purified protein goes through various steps of processing; hence it is possible that the protein may contain surfactants or other additives that may interfere with droplet formation. For such samples, using the 5 mm pathlength quartz cell is highly recommended. Figure 1. Analyte selection window for double stranded DNA quantitation Figure 2. Analyte selection window for non labeled protein quantitation Figure 3. Representative data for DNA quantitation Figure 4. Reproducibility data for DNA quantitation Figure 5. Representative data for protein quantitation Figure 6. Reproducibility data for protein quantitation