This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.Ĭell cloning Cloning unicellular organisms Ĭloning cell-line colonies using cloning ringsĬloning a cell means to derive a population of cells from a single cell. Further investigation of the resulting colonies must be required to confirm that cloning was successful. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Additionally, the cloning vectors may contain colour selection markers, which provide blue/white screening (alpha-factor complementation) on X-gal medium. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Finally, the transfected cells are cultured. A number of alternative techniques are available, such as chemical sensitisation of cells, electroporation, optical injection and biolistics. Following ligation, the vector with the insert of interest is transfected into cells. The vector (which is frequently circular) is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector (piece of DNA). Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size.
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