cell bio lab 6

Lab 6: Genomic DNA and Total RNA Extraction (Demo)

• Lab Objectives (Genomic DNA):

  • Understand genomic DNA extraction principles from plant tissue.
  • Extract genomic DNA from Arabidopsis thaliana leaf samples.
  • Measure and assess the purity of extracted genomic DNA.

• Lab Objectives (Total RNA):

  • Learn precautions for working with RNA.
  • Understand RNA extraction principles.

DNA Extraction Principles (Plants)

• Tissue Preparation: Grind tissue in lysis buffer to disrupt cell walls and membranes.
• Separation: Centrifuge to separate DNA from the organic phase (containing lipids and proteins).
• Precipitate & Wash: Add isopropanol to precipitate DNA, then wash with ethanol to remove impurities.
• Solubilize: Add water to solubilize the DNA.

Extraction Buffer Components

• Tris-HCl (1.50 mM): Maintains optimal pH for DNA extraction.
• EDTA (2.10 mM): Binds metal ions (Mg²⁺, Ca²⁺), preventing DNase degradation of DNA.
• NaCl (3.100 mM): Dissolves DNA and maintains stability.
• SDS (1.0%): Denatures proteins, disrupts cell membranes, releasing DNA.
• β-mercaptoethanol (5.10 mM): Strong reducing agent that breaks disulfide bonds in proteins, removing protein contamination.

Potassium Acetate and Isopropanol/Ethanol

• Potassium Acetate: Creates a high-salt environment to neutralize lysis solution pH, facilitating precipitation of SDS-bound proteins and cellular debris.
• Isopropanol/Ethanol: Facilitates interaction of K⁺ or Na⁺ ions with phosphate groups on DNA, reducing DNA solubility in water and causing precipitation.

DNA Quantification & Qualification

• NanoDrop Spectrophotometer: Measures DNA concentration and purity.
• A260/A280 Ratio: Indicates DNA purity:
  • Pure DNA: Ratio of 1.8-2.0
  • Lower ratios indicate contamination (residual phenol, sugar, or reagents).
  • Nucleic acids absorb at 260nm, proteins at 280nm.

RNA Precautions

• Storage: Store RNA samples at -80°C for long-term preservation. During use keep RNA samples on ice to maintain integrity.
• RNase Control: Control RNase sources, use RNase-free pipettes, tubes and clean all work surfaces.
• Safety: Wear gloves, masks and lab coats for protection.
• DEPC Water: Use DEPC water to inactivate RNase.

RNA Extraction Principle (Plants)

• Lysing: Extract cellular RNA.
• Filtering: Remove cell debris.
• Binding: Bind RNA to a specific medium.
• Washing: Remove impurities.
• Eluting: Release the purified RNA using RNase-free water.

RNA Assessment

• Assessment methods: Visual inspection using gel electrophoresis, quantification with spectrophotometry methods to assess quality.

Lab 6 Report Requirements

• Introduction: Explain the principle and purpose of genomic DNA extraction.
• Materials: List all chemicals and instruments, including concentrations.
• Methods: Describe the experimental steps performed.
• Results: Report extracted genomic DNA concentration, A260/A280 ratio, and unexpected findings.
• Discussion/Conclusion: Interpret results and discuss potential improvements; give significance of the lab work.