In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into the host organism, (6) Selection
Processes of recombinant DNA technology involve isolation of DNA of the desired DNA fragment. In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules. The DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides, and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus). The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease.
