Question

Describe the steps involved in plasmid isolation. Emphasize the purpose of each step, not the names and volumes of reagents used

Answer 1

Three main steps in the isolation of plasmid:

1. Growth of bacterial cells
2. Harvest and lysis of cells
3. Purification of plasmid

• First of all, bacterial cultures are grown overnight and then pelleted out. This pelleting of cels, gives the cellular material in the pellet. From this pellet plasmid will be isolated.
• Cells are suspended in alkaline lysis buffer, which contains EDTA. This chelates the magnesium ions in the DNA and prevents the nuclease activity.Lysozyme is added to break the bacterial cell wall and RNase is added to degrade RNA.
• SDS breaks the lipid of cell membrane and also proteins are denatured. So that the plasmid DNA preparation is free from RNA and protein contamination.
• Potassium acetate is added , which precipitates, all the dissolved proteins and other debris. And the mixture is centrifuged.
• Now, pellet containing debris is discarded and supernatant is retained.
• To purify plasmid DNA, phenol , chloroform and isoamyl alcohol are added.
• Phenol helps in dissolving the leftover proteins and chloform dissolves phenol.
• Again centrifuged and pellet containing plasmid is washed in ethanol, and dissolved in TE buffer.