Cloning-Isolation of Sequencing Grade Plasmid Virtual Lab Simulation

General Aim

To isolate high quality sequencing grade plasmid DNA.

Method

SDS/alkaline lysis.

Learning Objectives (ILO’s)

• To Apply the steps of high-quality DNA isolation.

• To understand the role of the devices, the reagents, and the enzymes used in the process of DNA extraction.

• To implement proper storage of samples.

Theoretical Background/Context

Highly purified plasmid DNA from Escherichia coli are required to obtain reliable sequencing results. This purification is based on the differential denaturation of chromosomal and plasmid DNA. 

Lysis solution containing sodium dodecyl sulfate (SDS) and sodium hydroxide denatures both chromosomal and plasmid DNA in bacterial culture. Neutralization with potassium acetate allows only plasmid DNA to reanneal and stay solubilized. Most of the chromosomal DNA and proteins precipitate in a complex formed with potassium and SDS.

 

Principle of Work

Cells are subjected to SDS/alkaline lysis, to break down the cell wall and extract DNA into the solution. The resulting lysate is neutralized and then centrifuged to pellet out cell debris and the SDS precipitate. The lysate containing the plasmid DNA is applied to the spin column containing the silica membrane, upon which the plasmid DNA will bind. After washing of the silica membrane to remove any contaminants, the plasmid DNA is eluted in a small volume of buffer.