DNA & RNA Quantification by Spectrophotometer.pptx

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1. Techniques in Biotechnology… M.PHOOL BADSHAH

2. Lecture-7 DNA/RNA Quantification by Spectrophotometer

3. Introduction A first step in many molecular biology investigations is quantifying the quantity of DNA and RNA. An approach that is frequently used to measure nucleic acid concentrations is UV-visible Spectrophotometry. Here's a basic rundown of how a spectrophotometer can be used to measure DNA and RNA:

4. What is Beer’s Law? Beer’s law was stated by August Beer which states that concentration and absorbance are directly proportional to each other. What is Lambert Law? Johann Heinrich Lambert stated Lambert law. It states that absorbance and path length are directly proportional.

5. Equipments and Reagents Nucleic Acid: DNA or RNA samples in solution are known as nucleic acid samples. UV-Visible Spectrophotometer: A device that detects the absorbance of light at certain wavelengths. Cuvettes or Microvolume Plates: Containers for holding the sample during measurement.

6. Procedure: 1- Sample Preparation: Use a blank solution (buffer only) for baseline correction and make sure your DNA or RNA sample is dissolved in an appropriate buffer or solvent. 2- Blank Measurement: Put a cuvette into the spectrophotometer that is solely filled with buffer. Using the blank solution, set the spectrophotometer to zero absorbance. 3- Sample Measurement: Pipette a tiny volume of your DNA or RNA sample into a cuvette or Microvolume plate (usually 1-2 μ L for microvolume instruments, 200-1000 μ L for normal cuvettes). Insert the microvolume plate or cuvette into the spectrophotometer.

7. 4- Wavelength Selection: Measure absorbance at 260 nm (A260) for DNA. Measure the absorbance of RNA at 260nm. 5- Absorbance Reading: After selecting the wavelengths, note the absorbance values. 6- Calculation of Concentration: Using the Beer-Lambert Law, determine the concentration of DNA or RNA based on the absorbance values: For DNA: Concentration (ng/ μ L)=A260×Dilution Factor×50 For RNA: Concentration (ng/ μ L)=A260×Dilution Factor×40 Any dilution or concentration phases in the sample preparation process are taken into account by the dilution factor.

8. 7- Purity Assessment: Assess the purity of DNA or RNA by determining the A260/A280 ratio. A pure sample usually has an RNA to DNA ratio of about 2.0 and a DNA ratio of about 1.8. Note: While the absorbance at 280 nm indicates the presence of proteins, the absorbance at 260 nm is specific to nucleic acids and is utilized for measurement. Verify the path length of the spectrophotometer, which is typically 1 cm for cuvettes, and modify computations as necessary. Make that the cuvettes or microvolume plates are clean and that the spectrophotometer is calibrated correctly.

9. Contaminants may cause RNA samples to absorb more light at 230 nm. You can measure A230 to determine purity if necessary. Microvolume measurements are frequently performed using nanodrop spectrophotometers, which enable small sample volumes and minimize sample consumption.