Overview
This lecture explains the steps for isolating plasmid DNA from bacterial cells using the alkaline lysis method, highlighting key reagents, the rationale for each step, and ways to assess DNA quality.
Introduction to Plasmid DNA Isolation
- Plasmid DNA is essential for gene cloning, amplification, and storage in molecular biology.
- Two main isolation methods: column-based (affinity chromatography, expensive) and alkaline lysis (chemical, inexpensive, efficient).
Resuspension of Bacterial Cells
- First step: Resuspend bacterial cells in resuspension buffer (Solution 1).
- Resuspension buffer contains Tris, EDTA, and glucose.
- EDTA chelates divalent ions (Ca²⁺, Mg²⁺) to inhibit DNases and destabilize membranes.
- Glucose maintains osmolarity to prevent premature cell lysis.
Alkaline Lysis Step
- Add lysis buffer (Solution 2) containing sodium hydroxide (NaOH) and SDS (sodium dodecyl sulfate).
- NaOH disrupts cell walls; SDS disrupts cell membranes, releasing DNA and proteins.
- The high pH denatures DNA, disrupting hydrogen bonds between base pairs.
Neutralization and Precipitation
- Neutralize with potassium acetate and acetic acid (Solution 3).
- Neutralization forms insoluble potassium dodecyl sulfate, which precipitates cell debris and proteins.
- Precise timing is required to allow plasmid DNA to renature but not chromosomal DNA.
- Centrifuge to pellet precipitated material; plasmid DNA remains in the supernatant.
DNA Precipitation and Purification
- Add isopropanol to the supernatant to precipitate plasmid DNA.
- Isopropanol reduces DNA solubility, facilitating precipitation.
- Wash the DNA pellet with ethanol to remove impurities, then dry and resuspend in nuclease-free water or Tris buffer.
- Store purified plasmid DNA at -20°C or -80°C.
Assessing DNA Quality
- Check plasmid DNA on an agarose gel; expect several bands (supercoiled, nicked, linear forms).
- The supercoiled form should be the most prominent band.
- Measure DNA yield via absorbance at 260 nm; a good yield is >500 ng/µL.
- The A260/A280 absorbance ratio should be ~1.8 for pure DNA.
Key Terms & Definitions
- Plasmid — Small, circular DNA molecule in bacteria, separate from chromosomal DNA.
- Alkaline Lysis — A method using high pH and detergents to break open cells and release plasmid DNA.
- EDTA — A chemical that chelates divalent metal ions, inhibiting DNase activity.
- SDS (Sodium Dodecyl Sulfate) — A detergent that disrupts cell membranes.
- Supercoiled DNA — The compact, twisted form of plasmid DNA, migrates fastest on a gel.
Action Items / Next Steps
- Review the full protocol and reagent preparation for each solution.
- Practice performing plasmid isolation and gel electrophoresis.
- Assess DNA purity and concentration using absorbance and gel analysis.