Transfection protocol
Transfection protocols are used in biological laboratories for introduction of foreign DNA, RNA, or protein molecules into cells of a eukaryotic nature. Transient or stable transfection protocols are often utilized. Transient transfection means that the gene will only be expressed transiently, or in other words, for a brief period of time. This method is used to test how gene modification will affect the function of certain gene(s).
Transfection is the method of delivering nucleic acids into cells by non-viral means. There are various ways to perform this type of gene transfer technology; by means of chemical, viral, or physical methods, providing valuable tool in studying gene function within the context of a cell.
Generally speaking, transfection is a tool which may be used to introduce charged DNA and RNA molecules into cells, which have a negatively charged membrane. Certain chemical reagents such as calcium phosphate and lipids bind to DNA (or RNA) and deliver transfection complex via cellular membrane.
Use of physical methods such as electroporation and microinjection allows direct transfect through the membrane of the cell and introduce the DNA (or siRNA), straight into the cytoplasm. Reporter technology has been also developed, and in combination with transfection reagents allows to perform various cell-based assays for testing of novel pharmaceuticals in vitro.
The procedure of isolating a certain DNA sequence and modifying the sequence for in vitro expression is known as molecular cloning. This type of cloning is regularly made use of to amplify DNA fragments which contain (coding) genes. However it may also be used to amplify DNA sequences such as randomly fragmented DNA, promoters and non-coding sequences. Molecular cloning is used in many biological experiments, and has a large array of technological applications, particularly in the field of large scale production of proteins.
Modern molecular cloning vectors have selectable markers (antibiotic), which allows only the cells with integrated vector to grow. Other features of specialized cloning vectors include sequences which allow for tagging, protein expression, DNA and single stranded RNA production.
