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Proteomics

Quantitative Estimation of DNA and RNA

Quantitative estimation of DNA and RNA

Estimation of nucleotides is an important step after sampleisolation to find out the amount of the nucleotide present and tocheck for the suitability of the sample for further analysis.

Learning Objectives:

After interacting with this learning object, the learner will be able to:

• Define the mechanism of detection.

• Operate the steps used in colorimetry.

• Assess the troubleshooting steps involved in the experiments.• Infer the law governing the colorimetric analysis.

• Describe the presence of DNA/RNA in the sample.

Note: The current IDD exists in two modes- interactive and automatic.Students taking lab course should select interactive (set as default),while the automatic mode may be selected for general users.

Gel Casting


Clean the surface of balance. Place a butter paperand tare its weight.

Gel Casting


Ethylenediamine tetra acetic acid (EDTA) stocksolution must to be prepared well in advance. EDTAdissolves only at pH 8. To measure the pH, go throughthe IDD for buffer preparation to use pH meter.

Gel Casting


The stock solution of TAE (50X) can be stored at roomtemperature and pH adjustment is not required.Prepare 1X TAE buffer which can be used for DNAquantification.

Gel Casting


The stock solution of TBE buffer (5X) can be stored atroom temperature and pH adjustment is not required.Prepare 1X TBE buffer, which can be used for RNAquantification.

Gel Casting


Agarose, a polysaccharide is used to separate andvisualize DNA/RNA. When heated, agarose melts andforms a polymer gel upon cooling. Weigh requiredvolume of agarose for casting the gel and addethidium bromide to it.

Gel Casting


Ethidium Bromide is a fluorescent dye and interactswith base pairs of DNA/RNA thereby forming acomplex. Ethidium bromide has UV absorbancemaxima at 300 and 360 nm, and an emission maximumat 590 nm.

Gel Casting


Place the gel casting unit, and place the appropriatecomb with the number of wells depending on thesamples to be loaded. Pour the gel solution into theunit. Take enough care to avoid air bubbles duringpouring the gel.

Making the Standards and Sample


Add running buffer to the unit with the requiredconcentration and place the gel in the tank.



The dye and the sample/standard must be mixed in a proper ratio beforeloading into the gel. The mixing step can be done on parafilm or on eppendorftube cap. The first well usually contains marker, with the immediate next wellcontaining the standards followed by the samples in the remaining wells.

Agarose Gel Electrophoresis


Set the required voltage and check the buffer levelbefore starting the run.

Agarose Gel Electrophoresis


After bands have been resolved in the gel, the gel canbe taken for visualizing DNA/RNA in UV transilluminator.

UV Transilluminator


Scan the image at required parameters settings. It isrecommended that the user saves a copy of thescanned image for use later.

UV Transilluminator


The analysis on the scanned image of the samplebands can be quantified by comparing with thestandards. If the sample bands run along the standardbands and are seen at the same position, it indicatesthe presence of DNA/RNA in the sample. If there areno bands within the marker run length, it indicatesthat DNA/RNA is not present in the sample.



Sample for the quantification of DNA is taken out.After diluting with buffer, vortex the solution for abrief period to ensure proper mixing. The solution isnow ready for the spectrometric reading.

Spectrophotometer


UV-Visible spectrophotometer has a monochromator,light source, sample holder and detector. Light fromthe source are converted to a monochromatic light ofparticular wavelength and allow it pass through thesample and amount of light that emerges is detectedby a detector.

Spectrophotometer


Absorbance of a sample is given by logarithmic ratio ofincident light to reflected light which is equal to theproduct of path length, epsilon constant calledcoefficient of absorbance and concentration of thesolution. From the above equation, one can calculatethe concentration of solution; if intensity of theincident light, reflected light and path length valuesare known. This equation is called Beer- Lambert’slaw.

Spectrophotometer


Auto zero and callibrate the instrument using thecontrol solution ( 1X TAE).

Spectrophotometer


Set the instrument at 260nm to set it at auto zerowith buffer and taking the reading for the samesample at 260nm. Repeat the step at 280nm and takethe ratio of 260/280.

Calculate the Concentration


A ratio value between 1.8 - 2.0 denotes presence ofnucleic acids.

A ratio value lower than 1.8 denotes presence ofproteins and/or other UV absorbers.

A ratio value higher than 2.0 indicates that thesamples may be contaminated with chloroform orphenol.

In either case (lesser than s1.8 or greater than 2.0) itis advisable to re-precipitate the DNA.

Calculate the Concentration


DNA concentration can be calculated using thefollowing formula.