The overall goal of this procedure is to determine the concentrations of nucleic acids and proteins accurately and reproducibly using a micro volume spectrophotometer. This is accomplished by first selecting the appropriate path length for the sample to be measured. The second step of the procedure is to measure a blank.
Next, the DNA or protein sample is measured. The final step of the procedure is to save the data in PDF or CSV format. Ultimately, highly reproducible results for DNA and protein.
Quantitation can be obtained rapidly and accurately by this method. The main advantage of this technique is it's fast, precise, and reproducible. To begin the procedure for DNA Quantitation, turn on the BIOS spec nano spectrophotometer.
Open the BIOS spec nano software from the PC desktop. Click on the simple nucleic acid D-S-D-N-A tab. The software will establish communication with the instrument and run an initialization sequence.
This verifies that the instrument is performing according to specifications. Next, select the path length. On the path length slider.
Three options are available for microliter volume samples. 0.2 millimeters or 0.7 millimeters can be used. And for dilute samples, there is an option to use a five millimeter path length vete.
In this demonstration, 0.7 millimeters is selected for measuring a two microliter sample. All calculations are based on the path length selected. Next in the software, click the setup tab and select auto wiping setup.
Select one for one wipe. Prior to sample measurement, or every time a new path length is selected, a blank measurement is required For a 0.7 millimeter path length pipette two microliters of water or buffer onto the pedestal. Click blank for blank measurement.
The upper window will move to make contact with the sample droplet and xenon flashes can be observed. When measurement is complete, the wiper will automatically wipe off the sample. Now the sample can be measured.
It is important to use a homogeneous sample solution as presence of particulate undissolved. Sample particle will result in irreproducible data. To avoid introducing air bubbles, carefully aspirate two microliters of the sample and then pipette the sample out uniformly from the pipette tip onto the pedestal.
Push the start button on the instrument. As before the upper window will move to make contact with the sample droplet and xenon flashes can be observed to determine reproducibility repeat measurement of the same sample three times all data recorded is automatically saved as A BUD file. To save as a PDF or CCSV file, click save P-D-F-C-S-V.
Results can also be saved as a CSV file by using the appropriate selection in the save tab. Protein quantitation can be done using the absorbance two 80 method. For protein quantitation, select the protein quantitation tab.
Enter the extinction coefficient epsilon two 80 if known, if Epsilon two 80 is not known, calculate it using Epsilon two 80 calc select 0.2 millimeters on the path length slider to measure a three microliter sample. Next in the software, click the setup tab and select auto wiping setup. Select two for two wipes to prevent any sample carryover and to ensure an accurate reading.
To measure a blank pipette three microliters of water or buffer onto the pedestal, click blank for blank measurement. Next, carefully pipette three microliters of protein sample onto the pedestal. Without introducing any air bubbles to begin measuring the sample, click start in the software or push the start button on the instrument.
Repeat measurement of the same sample three times to determine reproducibility shown. Here are data from measurements on a typical double stranded DNA sample. The formula used for calculating DNA concentration is sample concentration equals DF times the quantity of OD two 60 minus OD three 20 times NACF, where DF equals sample dilution factor and NACF equals nucleic acid concentration factor.
The nucleic acid concentration factor is set in accordance with the analyte selected. The calculated nucleic acid concentration and OD 2 62 80 ratio are indicated. This table contains reproducibility test results for DNA quantitation.
Together the data show results that are highly reproducible as evident from the low relative standard deviation of 0.26%Representative results from a protein concentration analysis for BSA are shown here. The microgram per milliliter protein concentration is based on the Epsilon two 80 value. Finally, this table shows a reproducibility analysis of the protein quantitation.
The relative standard deviation of 0.51%indicates that the BIOS spec nano measures od accurately not only for DNA, but also for proteins. Once mastered, multiple samples can be analyzed in one minute.
