Plasmid DNA Isolation - Cloning

Theoretical Background/Context

• Highly purified plasmid DNA from Escherichia coli is required to obtain reliable sequencing results through how to isolate plasmid DNA procedures.

• 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 through plasmid DNA isolation.

• 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 the silica membrane to remove any contaminants, the plasmid DNA is eluted in a small volume of buffer, and techniques such as how to concentrate DNA after miniprep can be applied if needed.

• Proper plasmid DNA storage conditions must be maintained, with the appropriate plasmid DNA storage temperature being critical to preserve DNA quality.