8 - Alkaline Lysis Plasmid Isolation

Overview of alkaline lysis procedure

1. Overnight culture and cell harvest

2. Suspension of bacteria in solution 1

3. Lysis of cells and denaturation of DNA in solution II

4. Addition of solution III neutralizes the pH. Plasmid DNA renatures while cellular proteins and genomic DNA precipitates

5. Separation of plasmid supernatant from precipitant

6. Further purification of plasmid DNA by adsorption chromatography

Use of centrifugation for culture

Bacteria are harvested by centrifugation

What happens if culture media remains throughout the alkaline lysis procedure

Residual media cell wall components present in the media may carry through the procedure and result in restriciton digest resistant DNA

Use of sodium chloride Tris EDTA (STE) solution in cell harvest step

Washing in STE may be used to ensure all traces of cell media are removed

Components of Solution I in alkaline lysis

50 mmol/L glucose, 25 mmol/L Tris-Cl (pH 8.0), 10 mmol/L EDTA (pH 8.0)

Use of glucose in alkaline lysis solution 1

Maintains osmotic pressure to prevent cell swelling/shrinkage

Use of EDTA in alkaline lysis solution 1

Binds divalent cations (Mg2 and Ca2+) required by DNAse I. Additionally, these cations are integral to the integrity of the cell membrane

Use of Tris-Cl in alkaline lysis solution 1

Biological buffer, maintains pH

Tris to replace cation in the cell membrane resulting in the release of lipopolysaccharide

Components of alkaline lysis Solution II

0.2N NaoH, 1% (w/v) sodium dodecyl sulfate

Use of NaOH in alkaline lysis solution II

Disrupts bacterial cell wall and membrane by hydrolyzing lipids and proteins. Alkaline pH denatures proteins by breaking hydrogen bonds

Use of SDS in alkaline lysis solution II

Dissolves the plasma membrane releasing cellular proteins and nucleic acids.

Forms complexes with cellular proteins and genomic DNA (preventing the DNA from coming back together)

Components of alkaline lysis solution III

3 mol/L potassium acetate, 11.5% v/v glacial acetic acid

Use of Potassium acetate in alkaline lysis solution III

Potassium forms a white flocculant, potassium dodecyl sulfate genomic DNA and denatured stick to the flocculant

Use of Acetic acid in alkaline lysis solution III

Neutralizes solution II, plasmid DNA preferentially renatures

Alkaline lysis adsorption chromatography

In the presence of a high salt buffer and a chaotropic agent, plasmid DNA adsorbs to silica. DNA is eluted off the membrane using a low salt buffer or water

Effect of residual media in alkaline lysis

Resistant to restriction digest

Effect of cell clumps in solution I in alkaline lysis

Decreased yield - cell clumps don’t all lyse, only outside of clump does

Effect of undermixing solution II in alkaline lysis

Decreased yield - NaOH and SDS don’t get to cells

Effect of prolonged time in solution II in alkaline lysis

• Single stranded plasmid

• Resistant to restriciton enzymes

Effect of overmixing solution II and III in alkaline lysis

Fragmentation of gDNA, causing gDNA contamination in final product

Effect of transfer of flocculant to the column in alkaline lysis

Blocked column, decreased yield and purity