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

Overview

  • Lysis solution (solution II) is used for the isolation of plasmid DNA by the alkaline lysis method.
  • The plasmid-containing bacterial cells are lysed by treatment with the lysis solution.
  • Lysis solution contains sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS).
  • SDS is a detergent that solubilizes the phospholipid and denatures proteins, leading to lysis and release of the cell contents. The denaturing action of SDS also releases protein from DNA, leaving the DNA (both genomic and plasmid DNA) free from proteins.
  • High alkaline condition due to NaOH denatures the plasmid and genomic DNA.

Requirements

Reagents and solutions
> 10 N Sodium hydroxide (NaOH) solution
> 10% sodium dodecyl sulfate (SDS)
> Deionized / Milli-Q water

Equipment and disposables
> Measuring cylinder
> Conical flask / Beaker

Composition
> 0.2 N Sodium hydroxide (NaOH)
> 1% (wt/vol) Sodium Dodecyl Sulfate (SDS)

Objective
Preparation of 10 ml of lysis solution (solution II)

Preparation

Step 1: To prepare, 10 ml of lysis solution, take 8.8 ml of Deionized / Milli-Q water in a 10 ml measuring cylinder.

Step 2: Add 0.2 ml of 10 N NaOH solution and 1.0 ml of 10% sodium dodecyl sulfate (SDS).

Note:
You may see some white precipitate when you add SDS. Dissolve it by mixing.

Precaution:
Do not mix concentrated stock solutions together. This will cause precipitation.

Storage
The solution can be stored at room temperature for a week. It is recommended to prepare a fresh lysis solution for optimal lysis.

Applications
Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare a lysis solution of various volumes.
Reagents / Volume10 ml25 ml50 ml100 ml
10 N Sodium hydroxide (NaOH)0.2 ml0.5 ml1 ml2 ml
10% sodium dodecyl sulfate (SDS)1.00 ml2.5 ml5 ml10 ml
Water8.8 ml22 ml44 ml88 ml

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