NanoDrop Microvolume Quantitation of Nucleic Acids

The NanoDrop Sample retention system is innovative technology that uses the inherent surface tension of liquids to hold and measure micro volume samples between two optical pedestals without the use of QTS or capillaries. As little as one microliter of sample is pipetted directly on top of the lower optical surface. Upon closing the spectrometer arm, a liquid column is created between the upper and lower optical pedestals.

By surface tension forming a vertical optical path, the vertical path length can automatically change in real time during the measurement. Shortening the path length enables the measurement of higher concentrations, effectively removing the need to perform dilutions for most nucleic acid samples. Furthermore, the sample retention system expedites the experimental process with a fast cleanup, which consists of simply wiping the optical surfaces with a laboratory Wipe.

Today we'll be demonstrating two procedures for determining nucleic acid concentration using thermos scientific micro volume instrumentation. The first protocol is a direct nucleic acid measurement using the NanoDrop 2000 C spectrophotometer. The second protocol is an indirect fluorescence nucleic acid measurement using the NanoDrop 3, 300 fluoro spectrometer.

To Begin Clean the upper and lower optical surfaces of the micro volume spectrophotometer sample retention system by pipetting two to three microliters of clean deionized water onto the lower optical surface. Close the lever arm ensuring that the upper pedestal comes in contact with the deionized water. Lift the lever arm and wipe off both optical surfaces with a clean, dry lint-free lab wipe.

Next, open the NanoDrop software and select the nucleic acid application. Use a small volume calibrated pipetter to perform a blank measurement by dispensing one microliter of buffer onto the lower optical surface. Lower the lever arm and select blank in the nucleic acid application.

Once the blank measurement is complete, clean both optical surfaces with a laboratory wipe. As before, choose the appropriate constant for the sample that is to be measured. Next, dispense one microliter of nucleic acid sample onto the lower optical pedestal and close the lever arm.

Because the measurement is volume independent, the sample only needs to bridge the gap between the two optical surfaces For a measurement to be made select measure in the application software, the software will automatically calculate the nucleic acid concentration and purity ratios. Following sample measurement review the spectral output. A typical nucleic acid sample will have a very characteristic profile.

Spectral indicators of a poor quality sample include wavelength chips in the trough and in the 260 nanometer peak. Common sources of contaminants associated with specific nucleic acid isolation techniques include phenol, triol, and column extraction. In the case of phenol triol extraction, residual reagent contamination may be indicated by abnormal spectra between 220 and 240 nanometers, as well as by shifts in the 260 to 280 nanometer region.

Conversely, residual guine from column extraction may contribute to a peak near 230 nanometers and a shift in the trough from 230 nanometers to approximately 240 nanometers. To accurately assess sample purity 2 62 80 and 2 62 30 ratio should be analyzed in combination with overall spectral quality. Pure nucleic acids typically yield a 2 62 80 ratio of approximately 1.8 and approximately 2.0 for DNA and RNA respectively.

This ratio is dependent on pH and ion strength of the buffer used to make the blank and sample measurements. Banana drop 3, 300 fluoro spectrometer employs the same patented micro volume sample retention system as the NanoDrop 2000 C spectrophotometer. With fluorescence measurements being performed with as little as one microliter of sample, the user chooses one of three LEDs as the excitation source, which fires at the sample horizontally.

The subsequent sample fluorescence is then collected at a 90 degree angle vertically into an internal spectrometer. To demonstrate high sensitivity micro volume nucleic acid quantitation using the NanoDrop 3, 300, A pico green fluorescence kit is used to begin equilibrate, the kit standards and all nucleic acid samples to room temperature. Fluorescent samples are light sensitive and should be kept in amber or aluminum wrapped tubes.

Prepare enough one XTE buffer for all standards and samples to be measured as well as for the pico green working solution that will be needed. Prepare one XTE buffer by diluting the provided 20 XTE buffer with nucleus free water per the manufacturer's protocol. Reaction volumes can range from 200 microliters to as little as 10 microliters.

In this example, we will prepare 10 microliter reaction volumes. Next, prepare serially diluted double stranded DNA standards and nuclease free vials or tubes. That will cover the expected concentration range of the unknown DNA samples.

Transfer five microliters of each of the diluted double stranded DNA standards into an individually labeled nuclease free amber or foil covered tube. Then aliquot five microliters of each double stranded DNA sample of interest into the appropriately light protected tube. After preparing the Pico Green Working solution according to the manufacturer's protocol, transfer an equal volume of the solution to each tube containing either double stranded DNA standard or double stranded DNA sample in this case five microliters.

Combine equal volumes of one XTE buffer and Pico Green working solution. To prepare the negative control which Serves as a reference solution. Mix the contents of each tube thoroughly by gentle pipetting and allow to incubate a room temperature for five minutes.

All standards and samples should be equilibrated to the same temperature before measurement as fluorescent signal is affected by temperature. To prepare the instrument and perform measurements first, clean the lower and upper surfaces of the sample retention system, pipette two to three microliters of deionized water onto the lower optical surface close. Then open the lever arm and blot the upper and lower pedestals with a clean lint-free laboratory wipe.

Ensure the surfaces are free of lint before proceeding. As lint can exhibit fluorescence in the presence of an excitation source and can interfere with the measurement. Open the instrument software.

Select the nucleic acids application and select the pico green double stranded DNA option pipette two microliters of one XTE to the lower pedestal. Close the lever arm and select blank in the instrument software field. Once the blank is complete, blot off the blank solution from both surfaces of the sample retention system.

Next, mix the reference solution by gentle pipetting using low retention tips. Pipette two microliters of the reference solution onto the lower pedestal and close the lever arm. Select reference under the standards tab and select the desired units.

Click measure to initiate the measurement Cycle. When the measurement cycle is complete, open the lever arm and thoroughly blot off the sample retention system surfaces. Measure up to five replicates of the reference using a fresh Eloqua for each replicate.

Repeat the process for the additional standards to build a standard curve. Up to seven standards may be used. The software is designed to store up to five replicates for each standard.

Replicates of the standards are averaged to generate a standard curve from which the sample concentrations are automatically determined. Once the standard curve is complete, select the samples tab, enter the respective sample ID information and measure each unknown sample. A valid curve indicates that the minimum number of standards has been met.

The DNA concentration is automatically based on The standard curve. NanoDrop Micro volume instrumentation can determine nucleic acid concentration by either direct A two 60 measurements or indirect fluoro metric methods. NanoDrop spectrophotometers can also be used to quantify proteins using Direct A two 80 absorbence or by protein color metric assays.