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Comprehensive Guide to DNA Extraction and Purification Techniques

Understand the principles, steps, and protocols involved in DNA extraction using lab equipment like automatic pipettes, vortex, and spectrophotometer. Learn how to measure DNA purity and concentration, and the applications of purified DNA in molecular biology research and diagnostics.

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Comprehensive Guide to DNA Extraction and Purification Techniques

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  1. DNA Extraction and Purification

  2. Lab Equipments Automatic pipettes Vortex Microcentrifuge Water bath UV-spectrophotometer

  3. Lab Equipments Cuvettes Eppendorf tube Tips Rack- test tube Rack- eppendorf tube

  4. DNA Extraction Principle: 1. Lysisof nucleated cells. 2. Removal of contaminants: Any substance other than DNA, e.g., proteins 3. Measurements:UV absorbance at 260nm and 280nm Purity of DNA solution: 260/280 ratio DNA concentration: Absorbance at 260nm

  5. DNA Extraction Steps: • Lysis of nucleated cells using lysis buffer. • Binding of DNA to the membrane of spin column. • Wash: using wash buffer. • Elution of pure DNA

  6. Spin ProtocolofDNA Purification from Blood

  7. Quantification of the purified DNA

  8. Measurements • Measure the Absorbance at 260nm. • Measure the Absorbance at 280nm.

  9. Measurements • Assess the DNA purity: 260/280 ratio (Accepted ratio: 1.7 - 1.9) • Calculate DNA Conc.: Provided A260 = 1.0, DNA is 50 μg/ml, unknown DNA Conc. can be calculated by cross multiplication A260 = 1.0 DNA conc. = 50 μg/ml A260 = 0.5 DNA conc. ? Note:-In case of diluting the eluted sample, multiplies the final concentration by the dilution factor. This can be adjusted by the spectrophotometer. Done by the spectrophotometer

  10. DNA yield DNA Yield = DNA Volume x final DNA Conc. Example: If you have Volume of DNA solution: 200μl (0.2 ml) Final DNA Conc.: 30 μg/ml Then, the yield (μg) = 0.2 ml x 30 μg/ml = 6.0 μg

  11. DNA Applications Purified DNA can be used for: 1. Molecular diagnosis of diseases. (e.g., sickle cell anemia) 2. Forensic applications. (e.g., paternity testing) 3. Molecular biology research.

  12. Molecular techniques using purified DNA: a. Amplification techniques: Polymerase Chain Reaction (PCR). b. Southern blotting. c. Restriction Fragment length polymorphism (RFLP).

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