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View additional product information for NanoDrop™ Lite Spectrophotometer - FAQs (ND-LITE)
51 product FAQs found
The NanoDrop Lite Spectrophotometer measurement pedestals are compatible with most solvents typically used in a life science laboratory. Wipe dilute acids from the pedestal surfaces immediately after a measurement is complete to ensure the pedestal surfaces stay conditioned.
Only hydrofluoric acid (HF), in any form, will damage the pedestal by dissolving the quartz fiber optic cable. Do not use hydrofluoric acid on the pedestal. Refer to the technical document, "Solvent Compatibility and Utility" for additional information:
https://assets.thermofisher.com/TFS-Assets/CAD/manuals/T045-NanoDrop-Instruments-Solvent-Compatibility.pdf
The LEDs in the NanoDrop Lite Spectrophotometer are only on during measurements.
The LEDs in the NanoDrop Lite Spectrophotometer are expected to last for the lifetime of the instrument.
We recommend confirming that your NanoDrop Lite Spectrophotometer is working within specifications every 6 months. The NanoDrop CF-1 Calibration Fluid is required to run the calibration check procedure. CF-1 is available from Thermo Fisher website (Cat. No. CHEM-CF-1)
The accuracy of your NanoDrop Lite Spectrophotometer can be checked by using the NanoDrop CF-1 Calibration Fluid to perform a calibration check. CF-1 is prepared from the NIST potassium dichromate standard SRM935 in acidified reagent grade water.
The NanoDrop Lite Spectrophotometer cannot be connected to a personal computer. The NanoDrop Lite Spectrophotometer is a local control instrument running preloaded applications
The NanoDrop Lite Spectrophotometer is a standalone unit with local control, which does not require a computer to operate. Data can be saved to a memory device and transferred to a computer.
Measurements for the NanoDrop Lite Spectrophotometer can be found in "Sample History" under the "Tools and Settings" menu to view or print previous measurements.
Data from each measurement made with the NanoDrop Lite Spectrophotometer is automatically saved in the instrument memory and can be transferred to a memory device at a later time. Data for up to 500 measurements are stored in the NanoDrop Lite memory. Once 500 measurements have been stored in memory, measurement #501 data will overwrite the measurement #1 data.
The sample output data from the NanoDrop Lite Spectrophotometer includes:
- sample number (auto-generated 1 through 500)
- time/date of measurement
- analyte being measured
- absorbance (A260 or A280)
- concentration
- 260/280 purity ratio (for nucleic acid measurements)
The NanoDrop Lite Spectrophotometer does not collect spectral data. It measures absorbance at three different wavelengths:
- A reference wavelength
- 260 nm
- 280 nm.
When using a NanoDrop Lite Spectrophotometer, negative absorbance values are most likely due to a blank measurement was made either using a solution with more absorbance than the sample buffer or on a dirty pedestal. We recommend cleaning the pedestal and making a new blank measurement with a fresh aliquot of the appropriate buffer.
When using a NanoDrop Lite Spectrophotometer, the blanking solution should always be the solvent or buffer used to dissolve the sample (at the same pH and ionic strength).
There are many reagents and organic solvents that could compromise the pedestal surface properties, causing samples to flatten out rather than bead up. Use the NanoDrop PR-1 reconditioning compound as a rapid means of reconditioning the pedestals when the pedestal surface properties have been compromised and samples spread out on the pedestal. NanoDrop PR-1 Reconditioning Kits are available through Thermo Fisher website (Cat. No. CHEM-PR1-KIT)
An application of water at the end of a measurement session is generally all that is necessary to keep the measure surfaces clean and conditioned.
If additional cleaning is required, refer to the technical document, Cleaning and Reconditioning for the NanoDrop Lite, for additional information:
https://assets.thermofisher.com/TFS-Assets/CAD/Product-Bulletins/2771-ND-CleaningRecondition-UG.pdf
CAUTION: Do not use detergents or isopropanol as cleaning agents as their use may result in the pedestals becoming unconditioned.
Yes, wiping with dry lint-free lab wipes remove samples very effectively. The highly polished quartz stainless steel surfaces of the sample retention system are resistant to sample adherence. However, if a sample is left to dry on the pedestal, more extensive cleaning is required.
Refer to the technical document, Cleaning and Reconditioning for the NanoDrop Lite, for additional information:
https://assets.thermofisher.com/TFS-Assets/CAD/Product-Bulletins/2771-ND-CleaningRecondition-UG.pdf
We do not recommend using the NanoDrop Lite Spectrophotometer for colorimetric methods.
The Protein A280 application of the NanoDrop Lite is designed for measuring purified proteins that absorb at 280 nm. Calculations are based upon Beer's Law, using a protein specific extinction coefficient. Colorimetric assays require standard curve generation and absorb light at wavelengths other than 280 nm.
If you are currently using a colorimetric assay to measure proteins, it is recommended that you use one of the preprogrammed colorimetric methods available on the NanoDrop 2000/2000c.
The dynamic range depends on the assay type selected for the protein being measured.
The following options are available:
- 1A/cm = 1 mg/mL
- IgG
- BSA
Refer to the "NanoDrop Lite User Guide" for more information on dynamic range and expected reproducibility:
https://assets.thermofisher.com/TFS-Assets/CAD/manuals/User-Guide-2752-NanoDrop-Lite-UG.pdf
Yes, we recommend prior purification of proteins because absorbance measurements will be affected by the presence of non-protein molecules which absorb at 280 nm. The NanoDrop Lite Spectrophotometer is not designed to measure non-purified proteins and it does not measure the A280/A260 purity ratio for proteins.
The dynamic range depends on the nucleic acid being measured. Refer to the "NanoDrop Lite User Guide" for more information on dynamic range and expected reproducibility:
https://assets.thermofisher.com/TFS-Assets/CAD/manuals/User-Guide-2752-NanoDrop-Lite-UG.pdf
Yes, we recommend performing purification prior to measurement with the NanoDrop Lite Spectrophotometer. Absorbance measurements are not specific for a particular sample type. Therefore, any analyte that absorbs at 260 nm (DNA, RNA or free nucleotides) will contribute to the total absorbance of the sample.
The NanoDrop Lite Spectrophotometer is designed to measure the absorbance and calculate the concentration of nucleic acids (260 nm) and purified proteins (280 nm). This includes dsDNA, ssDNA, RNA and purified proteins.
The NanoDrop Lite Spectrophotometer uses a 0.5 mm pathlength and all reported concentration results have taken into account the pathlength. The absorbance reported for all measurements is normalized to a 10 mm pathlength.
Your sample size should not affect your concentration results because, all calculations are volume independent. Sample concentrations for all applications are calculated using the Beer-Lambert equation, which relates concentration to absorbance using analyte and wavelength specific extinction coefficients or conversion factors.
Although 1 µL volumes are usually sufficient for most sample measurements, increasing the sample size to 2 µL will ensure proper column formation for samples with reduced surface tension properties.
Beer-Lambert Equation is as follows:
A = E * b * c
- A is the absorbance of the sample
- E is the wavelength-dependent molar absorptivity coefficient (or extinction coefficient) expressed in units of L/mol-cm
- b is the pathlength in cm
- c is the analyte concentration in mol/L or molarity (M).
For Nucleic Acid calculations, the Beer-Lambert equation is modified to use an extinction coefficient with units of ng-cm/mL. Using this extinction coefficient gives a manipulated equation:
c = (A * e)/b
- c is the nucleic acid concentration in ng/µL
- A is the absorbance of the sample
- e is the nucleic acid specific concentration factor in ng-cm/µL (dsDNA = 50 ng/µL; ssDNA = 33 ng/µL; RNA = 40 ng/µL; dsRNA = 40 ng/µL)
- b is the pathlength in cm
NanoDrop spectrophotometers use light pathlengths from 1.0 mm to as low as 0.05 mm (model dependent) to make measurements, allowing the quantification of samples up to 50 or 200 times more concentrated than traditional cuvette based spectrophotometers are able to measure. Applications such as Nucleic Acid and Protein A280 will display this absorbance as a 10 mm equivalent absorbance.
The sample concentrations are calculated according to Beer's Law which is volume independent. Beer's Law relates absorbance to concentration using a wavelength specific molar extinction coefficient or the use of a nucleic acid concentration factor and a pathlength of 1 cm.
The software is programmed to determine the best pathlength for the measurement based upon the absorbance signal of the sample at the analysis wavelength for the Nucleic Acids module, Protein A280 module, and Custom methods. On the other hand, the autopathlength function in the UV-Vis module monitors the entire wavelength range to optimize pathlength selection. The absorbance signal is normalized to a either 10 mm or 1.0 mm pathlength equivalent depending on module.
UV-Vis data is reported at a normalized 1.0 mm pathlength. As a result, absorbance values reported here will be reported 10-fold lower than those reported by the Nucleic Acid or Protein A280 applications.
Selecting autopathlength should be considered if the peak of interest is greater than 1.25 A at a 1.0 mm pathlength or to ensure that the entire sample absorbance spectrum remains within the linear response of the detector. With autopathlength deselected, the instrument uses only a 1.0 mm pathlength which is optimized for absorbance signal less than 1.25 A.
Both assays are mix-and-measure (minimal incubation times and no heating steps). Pierce 660 Assay has a more stable end-point. Pierce 660 reaction will not continue to progress and form aggregates.
When utilizing the measurement pedestal, NanoDrop spectrophotometers will report the Pierce 660 assay absorbance using a 1.0 mm pathlength. As a result, the absorbance displayed will be 10-fold lower than if measured in a conventional 10 mm cuvette. This reduction in signal does not compromise the sensitivity or linear range of the assay.
The Lowry module reports absorbance signal at a 1.0 mm pathlength rather than the conventional 10 mm pathlength resulting in a 10-fold reduction in absorbance signal. Consequently, the reduction in signal will not compromise the sensitivity or dynamic range of the assay. In the cases of extreme low signal, it is typically due to the formulation of the Lowry reagent from the specific vendors. We have had the greatest success with the Pierce product line of colorimetric assays.
General Information:
Wavelength (modified Lowry protocol for a NanoDrop spectrophotometer) is measured at 650 nm and normalized at 405 nm
Dynamic range of assay is 0.2 mg/mL to 4.0 mg/mL (BSA)
A poorly mixed Bradford assay will not properly complete and may form a gradient in the tube. This can affect reproducibility.
Some Bradford assay manufacturers' formulations will not provide sufficient absorbance to obtain an accurate and reproducible standard curve.
The Bradford reagent will form particulates as a reaction progresses. These particulates may cause light scatter during a sample measurement, resulting in higher than expected variation between measurements. Particulates can be minimized by limiting the incubation time to 10 min prior to measuring as per the Thermo Scientific Pierce protocol. In addition, a sample volume of 2 µL should be used for all colorimetric assay measurements.
To ensure accurate results, sample heating must be uniform for both standards and unknown samples.
When utilizing the measurement pedestal, NanoDrop spectrophotometers will report the BCA assay absorbance using a 1.0 mm pathlength. As a result, the absorbance displayed will be 10-fold lower than if measured in a conventional 10 mm cuvette. This reduction in signal does not compromise the sensitivity or linear range of the assay.
A purified protein should show a 260/280 purity ratio of approximately 0.63.
RIPA buffer produces a particularly strong absorbance signal at the 280 nm wavelength. As a result, it will either over estimate or under estimate protein concentrations and interfere with the protein purity ratio.
Protein samples in RIPA buffer should be quantified via the Pierce Protein 660 or BCA colorimetric assays using a full spectrum NanoDrop model.
Find additional tips, troubleshooting help, and resources within ourProtein Purification and Isolation Support Center.
The difference between the E1% and E0.1% values are the associated units for the results.
The E1% value has units of a 1% solution or 1 g/100mL. In the NanoDrop software this value is converted to an E0.1% value to display protein concentrations in mg/mL units.
Please refer to Tech Tip #6: Extinction Coefficients for further detail: https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0006-Extinction-coefficients.pdf
Protein concentrations obtained from the Protein A280 module are calculated using a 1% solution extinction coefficient (E1%) in place of the molar extinction coefficient. Each purified protein will have a unique E1% value which can be obtained from either references or, if produced commercially, from the vendor. The user may choose to use a known E1% value or input the molar extinction coefficient and molecular weight of the protein into the NanoDrop software to obtain the mg/mL protein concentrations.
If only a rough estimation is desired, the 1 Abs = 1 mg/mL can be used instead of a specific E1% value.
Pedestal surfaces may become "un-conditioned" and possibly inhibit proper liquid column formation required for measurement precision. Refer to this Cleaning and Reconditioning document for more information: http://tools.thermofisher.com/content/sfs/brochures/T005-NanoDrop%201000-&-NanoDrop%208000-Cleaning-and-Reconditioning.pdf
Measuring at or below the detection limit may result in SD values outside the precision specification. Refer to instrument user manual for detection limits for specific applications.
Evaporation can occur when samples remain on the pedestal beyond the time required to make one measurement, therefore we recommend always using a fresh aliquot for each measurement.
Poor sample homogeneity is commonly associated with non-homogeneous DNA samples. Due to the small sample volumes used for pedestal measurements it is important to mix the sample thoroughly. Samples containing large molecules such as genomic or lambda DNA are particularly susceptible to this phenomenon. If compatible with the protocol being used, heat the DNA samples to 63 degrees C and lightly finger vortex before measurement to ensure the nucleic acid is properly in solution.
NanoDrop CF-1 Calibration Fluid is the only photometric standard available to assess the accuracy of the NanoDrop spectrophotometer pedestal. If the control sample shows unexpected results the user should confirm the calibration of the instrument with the CF-1 fluid.
Some control examples include:
- Nucleic Acids: Fermentas GeneRuler Express DNA Ladder 100-5000 bp (Cat. No. SM1551)
- Protein: Pierce Bovine Serum Albumin Standard Ampules, 2 mg/mL (Cat. No. 23209)
The accuracy of NanoDrop spectrophotometers can be assessed by using NanoDrop CF-1 Calibration Fluid in conjunction with the calibration check application in the NanoDrop software.
Please review the following information regarding acceptable purity ratios, when using NanoDrop spectrophotometers:
- Nucleic acid samples: A260/A280 ratios of 1.8-2.2; A260/A230 ratios of 1.8-2.2 are generally considered pure
- Protein samples: A260/A280 ratio of approximately 0.63 is generally considered pure
Please review the following possible causes for 260/280 purity ratios to fall outside of the generally accepted range when using a NanoDrop spectrophotometer:
- The starting sample or the extraction method may result in contaminants in the final extract, e.g. guanidine (often from column based kits), phenol carryover or carbohydrates from the original cells (often a problem with plant samples). This is often best diagnosed by looking at the spectra, as many contaminants have characteristic profiles.
- Improper Blank: An incorrect or highly absorbent blanking solution or sample residue on the measurement pedestal may affect purity ratios.
- Sample Concentration: If the sample is nearing the lower detection limit of the instrument. Contributions from instrument noise may affect the shape of the spectrum. Check instrument specifications for lower detection limits. Purity ratios cannot be accurately calculated for samples close to the lower detection limits of the instrument. If a NanoDrop 2000c is being used, a cuvette may be used for samples at very low concentrations.