Classical gene cloning and identification

Initial enzyme-based fragmentation of intact genomic DNA (usually chromosomes isolated as described earlier in this chapter) so that it is broken down into manageable fragment sizes for further manipulation. Ideally all/most fragments will contain one gene. 

Integration of the various fragments generated into cloning vectors, which are themselves small DNA molecules capable of self-replication. Typically, these are plasmids or viral DNAs and the composite or engineered DNA molecules generated are called rDNA. 

Introduction of the vectors housing the DNA fragments into host cells. 

An essential feature of the cloning vector used is that it must be capable of self-replication in the cell into which it is introduced, which is usually E. coli. Two of the most commonly used types of vector in conjunction with E. coli are plasmids and bacteriophage X. Plasmids are circular extra-chromosomal DNA molecules, generally between 5000 and 350 0000 bp in length, that are found naturally in a wide range of bacteria. They generally house several 

The next stage of the cloning process entails the introduction of the engineered vector into E. coli cells. This can be achieved by a number of different means. One approach (called transformation) involves co-incubation of the plasmids and cells in a solution of calcium chloride, initially at 0 °C, with subsequent increase in temperature to 42 °C. This temperature shock facilitates entry of plasmids into some cells. 

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