My Scientific Blog - Research and Articles: January 2018

NANODROP SPECTROPHOTOMETRY
(Quantification of Nucleic Acids)

NanoDrop is a Spectrophotometer that measure 0.5- 2 ml samples with high accuracy and reproducibility. The NanoDrop model offers the convenience of both the NanoDrop patented sample retention technology and a traditional cuvette for sample measurements.

Principle: It operates on Beer’s law.

When monochromatic light (light of a specific wavelength) passes through a solution there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is, the more concentrated the specimen is, the less light is transmitted through it.

The sample retention system in NanoDrop Spectrophotometry employs surface tension to hold the sample in place between two optical fibers. This enables the measurement of very highly concentrated samples without the need for dilutions. Using this technology, the full spectrum (190 – 840 nm) NanoDrop Spectrophotometers have the capability to measure sample concentration up to 200 times more concentrated than samples measured using the standard cuvette.

Sample Retention Pedestal Measurements:

— A 1 - 2 µL sample is pipetted onto a measurement pedestal.

— A smaller, 0.5 µL volume sample, may be used for concentrated nucleic acid and protein A280 samples.

— A fiber optic cable (the receiving fiber) is embedded within this pedestal.

— A second fiber optic cable (the source fiber) is then brought into contact with the liquid sample causing the liquid to bridge the gap between the ends of the two fibers.

— A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array analyzes the light passing through the sample.

— The instrument is controlled by PC based software, and the data is stored in workbook files (*.twbk) on the PC.

Pedestal Sample Size Requirements: Although sample size is not critical, it is essential that a liquid column is formed when using the pedestal option so that the pathlength between the upper and lower measurement pedestals is bridged with sample. The dominant factor determining the surface tension of a droplet is the hydrogen bonding of the lattice of water molecules in solution. Generally, all additives (including protein, DNA, RNA, buffer salts and detergent-like molecules) can reduce the surface tension by interfering with the hydrogen bonding between water molecules. 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.

Sample volumes are sufficient to ensure reproducibility

Aqueous solutions of nucleic acids: 1 µL
Purified protein: 2 µL
Bradford, BCA, Lowry or Protein Pierce 660 nm assays: 2 µL
Microbial cell suspensions: 2 µL

Note: It is best to use a precision pipettor (0-2 µL) with precision tips to ensure that sufficient sample (1-2 µL) is delivered. Lower precision pipettors (0-10 µL and larger) are not as good at delivering 1 µL volumes to the measurement pedestal. If the user is unsure about the sample characteristics or pipettor accuracy, a 2 µL sample volume is recommended.

Pedestal Basic Use

Raise the sampling arm and pipette the sample onto the lower measurement pedestal.

Lower the sampling arm and initiate a spectral measurement using the software on the PC. The sample column is automatically drawn between the upper and lower pedestals and the measurement is made.
When the measurement is complete, raise the sampling arm and wipe the sample from both the upper and lower pedestals using a dry, lint-free laboratory wipe. Simple wiping prevents sample carryover in subsequent measurements for samples varying by more than 1000-fold in concentration.

Cuvette Measurements

The NanoDrop will accept 10 mm cuvettes up to 48 mm tall. When measuring samples using micro, semi-micro, or ultra-micro cuvettes, we recommend using masked cuvettes. Masked cuvettes ensure that all light hitting the detector has passed through the sample. Unmasked cuvettes can allow light to hit the detector that has not passed through the sample. Expected differences between unmasked plastic cuvettes can lead to significant measurement error, especially at low concentrations.

Cuvette Sample Size Requirements:

It is essential to ensure that the sample volume in the cuvette is adequate to allow the light to pass through a representative portion of the sample when making a measurement.

Cuvette Basic Use

Add the sample to the cuvette and ensure that the volume is sufficient to cover the light path.
Raise the sampling arm and insert the cuvette into the instrument. Insert the cuvette noting the direction of the light path indicated by the etched arrow.
The sampling arm must be lowered into the down position for cuvette measurements.
Initiate a spectral measurement using the software on the PC.
When the measurement is complete, remove the cuvette, rinse thoroughly and dry between samples.

Requirements:

Sample to be measured
NanoDrop
Micropipette with tips
Lint free lab wipes
Purified water
Blanking solution (D/W, TE, TB Tris or others depending on your sample)

Applications:

DNA Analysis (After extraction): Nucleic acid (DNA/RNA)

Qualitative analysis Quantitative analysis

Size-Quality Purity- Yield

Agarose gel electrophoresis Spectrophotometry

Why Quantify?

• To check concentration and purity of DNA/RNA present in the solution.

• Determine if samples are useful for downstream applications like: PCR, Restriction digests etc.

Advantages of the nanodrop

• It is a simple machine and economical on space.

• Easy-to-use spectrophotometer

• Can measure small volumes of DNA, RNA and protein concentrations.

NanoDrop Spectrophotometric measures

Measures DNA, RNA (A260) and Proteins (A280) concentrations and sample purity (260:280).

Absorbance at 260 nm

• Nucleic acids absorb UV light at 260 nm due to the aromatic base moieties within their structure. Purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine) both have peak absorbances at 260 nm, thus making it the standard for quantitating nucleic acid samples.

Absorbance at 280 nm

• The 280 nm absorbance is measured where proteins and phenolic compounds have a strong absorbance. Similarly, the aromaticity of phenol groups of organic compounds absorbs strongly near 280 nm.

Absorbance at 230 nm

• Many organic compounds have strong absorbances at around 225 nm. In addition to phenol, TRIzol, and chaotropic salts, the peptide bonds in proteins absorb light between 200 and 230 nm.

NanoDrop Spectrophometric measurements

• The A260/280 ratio shows the purity of the sample analyzed.

Nucleic Acid Type	Approximate A₂₆₀/A₂₈₀Ratio
Pure DNA	1.8
Pure RNA	2.0
Pure Protein	0.57

• A260/230 ratio indicates the presence of organic contaminants, such as (but not limited to): phenol, TRIzol, chaotropic salts and other aromatic compounds.

• Samples with 260/230 ratios below 1.8 are considered to have a significant amount of these contaminants

• Various nanodrop models are available e.g Nanodrop 8000, Nanodrop lite and Nanodrop 2000/2000c which is currently available at BecA labs.

Note that: Because of the low concentrations, sometimes it is difficult to assess the purity of the samples (esp. RNA) by analyzing the A260/280 and A260/230 ratios. Quantification cannot be assessed by the NanoDrop because they are outside the lowest concentrations the NanoDrop is designed to measure. A more sensitive method such as an Agilent Bioanalyzer or Qubit analysis is recommended.