Preparation of Neutralization Solution (Solution III) for Plasmid Isolation by Alkaline Lysis Method

Overview

  • The neutralization solution (solution III) is used for the isolation of plasmid DNA by the alkaline lysis method.
  • The neutralization solution is nothing but a potassium acetate solution which has pH 4.8.
  • The addition of the neutralization solution in lysed bacterial cells brings the pH back to normal, resulting in the precipitation of protein and genomic DNA.
  • Both plasmid and genomic DNA renatures upon the addition of the neutralization buffer. While plasmid DNA renatures in correct conformation due to its circular and covalent structure, therefore, remains in the solution, genomic DNA precipitates due to a random association of both of its strands.
  • Sodium dodecyl sulfate (SDS) of the lysis buffer reacts with Potassium acetate and forms insoluble Potassium dodecyl sulfate (KDS).

Requirements

Reagents and solutions
> 5 M Potassium acetate (CH3CO2K) solution
> Glacial acetic acid
> Deionized / Milli-Q water

Equipment and disposables
> Measuring cylinder
> Conical flask / Beaker

Composition
> 3 M Potassium
> 5 M Acetate

Objective
Preparation of 100 ml of Neutralization solution (solution III)

Preparation

Step 1: To prepare, 100 ml of Neutralization solution, take 28.5 ml of Deionized / Milli-Q water in a 100 ml measuring cylinder.

Step 2: Add 60 ml of 5 M Potassium acetate and 11.5 ml of glacial acetic acid. Mix the solution.

Storage
The solution can be stored at room temperature in a tightly-closed bottle for a year.

Applications
Plasmid isolation by alkaline lysis method

Follow the table To prepare a neutralization solution of various volumes.
Reagents / Volume10 ml25 ml50 ml100 ml
5 M Potassium acetate6.0 ml15 ml30 ml60 ml
Glacial acetate acid1.15 ml2.875 ml5.75 ml11.5 ml
Water2.85 ml7.13 ml14.25 ml28.5 ml

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