Step 5

Amplifying RAP6 Gene

Initially primers are designed for the RAP6 gene of interest and this process occurs through polymerase chain reaction (PCR), which is a scientific technique in molecular biology used to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Primers are molecules that serve as a starting material for a polymerization process and are commonly used in biochemistry. Primarily the denaturation process occurs in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound. Subsequently, the annealing process takes place where DNA or RNA pair to by hydrogen bonds to a complimentary sequence, forming a double-stranded polynucleotide; thus, referring to the binding of a primer to a DNA strand during a PCR. Thereafter, primer extension goes about and is a technique whereby the 5’ ends of RNA of DNA can be mapped and as a result, the RAP6 gene of interested is then amplified.

Recombinant DNA

Basically, this is the process of recombinant DNA or DNA that has been formed artificially by combining constituents from different organisms. The RAP6 constructs are simply DNA constructs that are an artificially constructed segment of nucleic acid that is going to be “transplanted” into a target tissue or cell. Hence these constructs contain a DNA insert, which contains the gene sequence encoding the protein of interest that has been sub cloned into a vector, which contains bacterial resistance genes for growth in bacteria, and promoters for expression in the organism. After that the transformation process occurs on the JM109 or bacterial cells and is simply the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous DNA from its surrounding and taken up through the cell membrane(s). In consequence, allowing DNA purification in which nucleic acid methods are the techniques used to study DNA and RNA, thus extracting DNA from a cell in a pure form.

Cell Culture

Ultimately, the 293T or mammalian cells are transfected in where the cells are infected with free nucleic acid by the introduction of genetic material in this way. Initially the purified DNA (siRNA) is added to the medium or buffer EC-R. Then the RNAiFect transfection reagent is added for specific intracellular delivery of reagents and hence mixed as well as incubated for about ten to fifteen minutes at room temperature. After the transfection reagent is applied to all the cells and again the cells are incubated until evaluation of silencing. Therefore, enabling intracellular distribution and observation of results through microscope immunofluorescence.