Cloning Process
The basic steps in a gene cloning experiment are as follows (see Figure 1). The first step is the polymerase chain reaction (PCR) process. PCR is a process of amplifies DNA in vitro to make a thousand cope of DNA. The insert gene was amplified by a specific forward and reverse primer, which are design for this gene. The product of PCR process was analyzed on agarose gel electrophoresis. The isolated DNA was purified and then fragmented with restriction enzyme BamHI and Hind III. the vector was cut by the same restriction enzyme. The insert DNA fragments were then incorporated into vector by ligation process. T4 DNA ligase was used to join the strands between the insert and the vector by catalyzing the formation of a phosphodiester bond. The next step was introducing the recombinant plasmid into bacterial host cell by transformation process. The cells were plated on agar medium, which contained ampicillin. Ampicillin is an antibiotic. They either kill off or prevent bacterial grow. Only the transformed cells, which contained insert gene, will growth. The non-transformed cells, which did not contain insert gene, will be killed off. Then the colony of cell containing the insert gene was isolated and screening. To screening the result of cloning, the digestion was performed by using the same restriction enzymes. Then the fragments were run on agarose gel electrophoresis to analyze the size of fragments. The result of the cloning process was successful. I got the band that had the size around 700bp, which was the same size of insert gene. The isolated DNA cloning will be use to study the function of their protein.