- 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.
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
> 0.2 N Sodium hydroxide (NaOH)
> 1% (wt/vol) Sodium Dodecyl Sulfate (SDS)
Preparation of 10 ml of lysis solution (solution II)
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).
You may see some white precipitate when you add SDS. Dissolve it by mixing.
Do not mix concentrated stock solutions together. This will cause precipitation.
The solution can be stored at room temperature for a week. It is recommended to prepare a fresh lysis solution for optimal lysis.
Preparation of plasmid DNA by alkaline lysis method
|Follow the table to prepare a lysis solution of various volumes.|
|Reagents / Volume||10 ml||25 ml||50 ml||100 ml|
|10 N Sodium hydroxide (NaOH)||0.2 ml||0.5 ml||1 ml||2 ml|
|10% sodium dodecyl sulfate (SDS)||1.00 ml||2.5 ml||5 ml||10 ml|
|Water||8.8 ml||22 ml||44 ml||88 ml|