List the step involved in recombinant DNA technology.

Explanation

(i) DNA is isolated from the cells by using different enzymes like lysozymes for bacterial cells, cellulase for plant cell, chitinase for fungal cells. The DNA molecules are treated with Proteases, RNAses to remove any proteins, RNA associated, so that DNA is obtained in fure form.
(ii) Cutting DNA at specific location.
(a) Fragmentation of DNA is carried out by incubating purified DNA molecules with restriction enzyme at optimal conditions of temperature and pH for that specific enzyme. (b) Agarose gel electrophoresis technique is employed to check the progression of restriction enzyme digestion. (c) The similar process is repeated with vector DNA.
(iii) Separation and isolation of DNA fragment. It is carried out by technique gel electrophoresis.
(a) DNA fragments being negatively charged, can be separated by forcing them to move towards anode under an electric field through a medium/matrix (agarose). DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. The smaller the fragment size, the farther it moves.
(b) DNA fragments can be visualised by staining DNA with ethidium bromide followed by exposure to UV radiations. Bright orange colour bands of DNA become prominent in the gel. The separated bands of DNA are cut out from gel and extracted from the gel piece. This step is known as elution.
(c) Purified DNA fragments are used for digested set of DNA fragments (lane 2 to 4). reconstructing recombinant DNA by joining them with cloning vectors.