Gene cloning procedures

Figure 1. Schematic representation of basic steps involved in a gene cloning procedure, cat, chloramphenicol transacetylase.

Wendland, J. 2003. PCR-based methods facilitate targeted gene manipulations and cloning procedures. Current Genetics 44,115-123. 

Cloning procedure (in bacteria) Recombinant plasmids transfected into bacteria Antibiotic resistance used to select bacteria with recombinant plasmids Blot growth plate and probe with P-DNA for gene, or with Tantibody for protein... 

Before starting the cloning procedure, it is also important to consider how expression of the inserted gene will be determined. If antibodies are not available with which to detect the encoded protein, it may be appropriate to label the protein with an expression tag, such as myc (24) or FLAG (25), provided such a tag does not... 

A novel cDNA/PCR strategy for cloning rare mRNA species employs another kind of adapter—primer that has dual functions one part as a PCR primer and the other as an adapter (22). The amplified cDNA products carry blunt ends as if linkers have been attached. Therefore the DNA molecules can be cloned in a way similar to the conventional linker-mediated cloning procedures mentioned earlier. For cloning unknown genes, the cDNA/PCR cloning procedure thus requires prior methylation to protect any internal restriction sites. 

---