A DNA library is screened to determine the presence of a gene and to clone out either a full-length or smaller version of the gene. There are many strategies for cloning from a library. Typically, the library is introduced into bacteria, the bacteria are plated under cloning conditions, replicate plates of the bacterial colonies are….

Recombinant proteins are expressed in bacteria, yeast, mammalian or in the baculovirus system. The gene of interest is transfected into the appropriate host together with regulatory elements that allow very high levels of production. Protein production may be intracellular, or may accumulate in the bacterial periplasmic space, or be secreted into the medium of cells…..

Protein, peptide or coding DNA sequences are searched against databases to determine uniqueness, related families of proteins, and to suggest characteristic properties of a protein.

Purified DNA is required to prepare DNA libraries or microarrays, to make probes, and to transfect genes. DNA is prepared from bacteria, yeast, tissue culture cells, tissues, or plants. Preparations can be genomic DNA or plasmids. The scale is from 1 microgram to 1 milligram.

Restriction endonucleases recognize and cut DNA at unique nucleotide sequences. Delineation of these cleavage sites provides a map of the DNA, produces characteristic sizes of the fragments as detected on an agarose gel, and allows for the preparation of pieces of DNA to be used for making new vector or plasmid constructs.

To unequivocally identify a gene or cDNA, the DNA is submitted for sequencing. Using overlapping portions of the DNA, a contiguous sequence is generated which can be used for data mining.

Subcloning is used to amplify a desired DNA molecule. After a piece of DNA or a gene is obtained, by synthetic synthesis, restriction digestion, joining to another DNA by a ligase, etc., it is introduced into competent bacteria and transformants expressing the new gene are identified. The bacteria are cloned on selection plates whereby only….

Specific RNA species in an unfractionated preparation can be measured by immobilizing a sample in a spot (Dot Blot) or in a manifold slot (Slot Blot). Detection is by a labeled DNA probe that hybridizes to the immobilized RNA. For the Dot Blot, quantitation is usually visual whereas the Slot Blot format is more easily….

In situ hybridization localizes the expression of a particular mRNA among different cells in a tissue or cell preparation. A cell preparation is fixed on a slide and the RNA contents probed with radiolabeled or fluorescent probe.