Alkaline lysis

EDTA binds Magnesium and Calcium ions which prevents a DNase from degrading plasmid DNA.

Tris Hydrochloride (HCl) is a buffer solution used to stabilize the pH and protect the integrity of the DNA.

[3] Tris-HCl is necessary due to the high pH environment that is established in order to lyse open the cells.

Sodium Hydroxide establishes an alkaline environment that disrupts the phospholipid bilayer and breaks hydrogen bonds between double-stranded chromosomal DNA, making it single-stranded.

Sodium Hydroxide and SDS detergent lyse the cell and release its cellular contents including chromosomal and plasmid DNA, into the solution.

Under neutral conditions, hydrogen bonds reform between complementary base pairs, joining single strands to double-stranded DNA.

[4] The remaining chromosomal DNA strands, denatured proteins, and added chemicals stick together and precipitate out.

Transformation involves the use of restriction enzymes to cut plasmids at specific locations to allow for insertion of a gene of interest.

The recombinant plasmid is then introduced into a bacterial cell where it can replicate independently, allowing the bacterium to express the gene of interest.

Application of this process is common in the medical field with the production of insulin, hormones, and growth factors.

Recombinant DNA of a functional copy of the defective gene can be incorporated into a plasmid host cell.