Extraction and Purification of Plasmid DNA by Alkaline Lysis Method

Hello everyone,

Today Iam here with another method for Isolation of DNA. Here I will be mainly focusing on Extraction and Purification of Plasmid DNA by Alkaline Lysis Method. 

A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. Scientists have taken advantage of plasmids to use them as tools to clone, transfer, and manipulate genes. Plasmids that are used experimentally for these purposes are called vectors. Hence there must be a process which help us to isolate one.The method which is used for Extraction and Purification of Plasmid DNA is Alkaline Lysis Method.

Alkaline lysis is the method of choice for isolating circular plasmid DNA, or even RNA, from bacterial cells. It is probably one of the most generally useful techniques as is a fast, reliable and relatively clean way to obtain DNA from cells. If necessary, DNA from an alkaline lysis prep can be further purified.

There are basically 3 steps involved 

1) Growth of bacterial culture

The procedure starts with the growth of the bacterial cell culture (E.Coli containing PUC-18 ) harboring your plasmid. St LB Medium containing 100mcg/ml of ampicillin.

2) Harvesting and Lysis of Bacteria

When sufficient growth has been achieved, the cells are pelleted by centrifugation to remove them from the growth medium. Lysis by any one treatment , non-ionic or ionic Detergents, Organic Solvents, alkali or heat. SDS is used to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single stranded DNA (ssDNA).

3)Neutralization

Addition of potassium acetate  returns decreases the alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. 

5. Cleaning and concentration

Now your plasmid DNA has been separated from the majority of the cell debris but is in a solution containing lots of salt, EDTA, RNase and residual cellular proteins and debris, so it’s not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA.

There are several ways to do this including phenol/chloroform extraction followed by ethanol precipitation and affinity chromotography-based methods using a support that preferentially binds to the plasmid DNA under certain conditions of salt or pH, but releases it under other conditions. 

But easiest method for purification is Agarose Gel Electrophoresis to seperate plasmid DNA .

-Dixy