DNA Quantification and Standard Curve PDF

University of Petra Faculty of Dentistry Cellular and Molecular Biology lab DNA Quantification and Standard Curve Dr. Duaa Quedan Molecular Biology lab for Dental student Introduction Determining DNA concentration ensures precise experimental outcomes. Quantifying DNA allows researchers to optimize...

University of Petra Faculty of Dentistry Cellular and Molecular Biology lab DNA Quantification and Standard Curve Dr. Duaa Quedan Molecular Biology lab for Dental student Introduction Determining DNA concentration ensures precise experimental outcomes. Quantifying DNA allows researchers to optimize the amounts of other reagents used in experiments. It helps identify potential issues such as degradation, contamination, or insufficient sample material before proceeding with downstream applications. Introduction Nucleic acids exhibit characteristic absorbance patterns at specific wavelengths, particularly around 260 nanometers (nm). Spectrophotometer is utilized to measure DNA concentration depending on nucleic acids absorbance to the UV light. This method provides a rapid, accurate, and non-destructive means of quantifying DNA. DNA quantification protocol 1.Prepare the DNA Sample: Start by preparing a DNA sample in a suitable solvent, such distilled water (in this experiment). Ensure the DNA sample is adequately dissolved and free from any contaminants that could interfere with the measurement. DNA quantification protocol 2. Blank Measurement: Before measuring the DNA sample, set the spectrophotometer to measure absorbance at 260 nanometers (nm). Perform a blank measurement by filling a cuvette with the chosen solvent (distilled water) and placing it in the spectrophotometer. This establishes a baseline absorbance value. DNA quantification protocol 3. Measure DNA Sample Absorbance: Once the baseline has been established, carefully transfer a small volume (2 µL of DNA into 198 µL distilled water for 100X dilution) to a clean cuvette or microcentrifuge tube. Wipe the outside of the cuvette or tube to remove any residual liquid. Prepare 3 different dilutions of your sample as follow: 100X 500X 1000X DNA quantification protocol 4. Insert Sample into Spectrophotometer: Place the cuvette containing the DNA sample into the spectrophotometer and ensure it is positioned correctly according to the instrument's instructions. DNA quantification protocol 5. Record Absorbance Reading: Take the absorbance reading at 260 nm wavelength. The spectrophotometer will provide a numeric value representing the absorbance of light by the DNA sample at this specific wavelength. If your Absorbance reading below 0.1 or above 1.0. Repeat the experiment with different dilution. DNA quantification protocol 6. Calculate DNA Concentration: Using the absorbance reading obtained in step 5, calculate the concentration of DNA in the sample using the Beer-Lambert law equation: A=ϵcl Where: A = Absorbance reading at 260 nm ϵ = the average extinction coefficient for double-stranded DNA is 0.020 (μg/ml)−1 cm−1 l = Path length of the cuvette (typically 1 cm) DNA concentration (µg/ml)=A×D​/ϵ×l D = Dilution factor (if the sample was diluted) DNA quantification protocol 7. Construct a standard curve Plot each data point on the graph according to its corresponding values on the X and Y axes. Fit a Line: your data should suggest a linear relationship that can be modeled by a mathematical function. Label you X and Y axes. DNA quantification protocol 8. Assess Sample Purity: Additionally, measure the absorbance of the DNA sample at 260 nm and 280 nm to assess sample purity. A ratio of absorbance at 260 nm to 280 nm (A260/A280) of around 1.8 indicates pure DNA. While a ratio above or below may suggest protein contamination or impurities. Activity Construct 3 dilutions for your DNA sample. Measure Absorbance at 260 and 280 nm. Plot your data in a graph and draw a standard curve with the equation. Estimate the purity of your DNA