4-1.2.4. Concentration of DNA samples

Concentration of DNA can be done using ethanol along with salts such as sodium acetate, potassium acetate etc. These salts provide metal ions like sodium ions (Na+), potassium ions (K+) which help in aggregation and hence precipitation of DNA molecules.

Advantage

It leaves short-chain and monomeric nucleic acid components in solution. Ribonucleotides produced by the ribonuclease treatment are separated from DNA.

4-1.3. Isolation and Purification of Plasmid DNA

1.  Growth of the bacterial cell.

2.  Harvesting and lysis of the bacteria.

3.  Purification of the plasmid DNA.

4-1.3.1. Growth of the bacterial cell

It involves growth of the bacterial cells in a media containing essential nutrients.

4-1.3.2. Harvest and lysis of bacteria

Lysis of bacteria results in the precipitation of DNA and cellular proteins. Addition of acetate-containing neutralization buffer results in the precipitation of large and less supercoiled chromosomal DNA and proteins leaving the small bacterial DNA plasmids in solution.

4-1.3.3. Purification of Plasmid DNA

This step is same for both plasmid and genomic but former involves an additional step i.e.the separation of plasmid DNA from the large bacterial chromosomal DNA.

4-1.3.3.1. Methods for separation of plasmid DNA

Separation of plasmid DNA is based on the several features like size and conformation of plasmid DNA and bacterial DNA. Plasmids are much smaller than the bacterial main chromosomes, the largest plasmids being only 8% of the size of the E. coli chromosome. The separation of small molecules (i.e. plasmids) from larger ones (i.e. bacterial chromosome) is based on the fact that plasmids and the bacterial chromosomes are circular but bacterial chromosomes break into linear fragments during the preparation of the cell extract resulting in separation of pure plasmids. The methods of separation of plasmid DNA are described as below-