Several Science Questions In the process of DNA cloning, a gen

In the process of DNA cloning, a gene to be replicated is first obtained by treating the DNA molecule with restriction endonucleases and ligases (DNA, 1999). A gene can be obtained as a single restriction fragment after digestion of a larger molecule with a specific restriction enzyme. In this case, the DNA fragment comes from a virus. The next step is to prepare plasmids such as those from E.coli. The plasmids are cut in a single position, converting the normally circular DNA into a single linear strand with 5'-GAT-3' sticky ends. This plasmid DNA is now mixed with the viral DNA and ligases, and various recombinant ligation products are obtained, which consist of circular plasmid DNA with the viral DNA inserted into the position where the restriction site originally was.

The recombinant plasmid is now reintroduced into E. Coli and the plasmid plus the inserted gene will be replicated and copies will be passed on to daughter bacterial cells during cell division (DNA, 1999). This will give rise to a colony of E. Coli bacteria in which each bacterium contains multiple copies of the viral gene. To separate recombinant bacteria from non-recombinant bacteria, they can be plated on ampicillin plates containing X-gal. With most cloning vectors, including pUC8, the vector carries the ampicillin resistant gene from pBR322 and a second gene called lacZ, which is part of the E. Coli gene for the enzyme beta-galactosidase. The remainder of the lacZ gene is locate

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). Question 3 Western blotting is used to determine the relative amounts of a specific protein present in a sample using specific primary antibodies (Molecular, 2004). Samples are prepared from tissues or cells by homogenization in buffers that protect the protein of interest from degradation. The sample is separated using SDS-PAGE and then transferred to a membrane for detection fo the proteins. The membrane is incubated with a generic protein (e.g. milk protein) to bind to any remaining sticky places on the membrane. A primary antibody is then added to the solution which is able to bind to its specific protein. A secondary antibody-enzyme conjugate, which recognizes the primary antibody, is then added to find locations where the primary antibody bound. After the first antibody is added, it is rinsed off, and a secondary anti-IgG antibody tagged with a radioactive or enzymatic label can be added to the film (Western, 2004). These will bind to the already-bound primary antibodies specific for the desired protein. A second rinse is used to remove any unbound anti-IgG antibodies, and the radioactive bands can be detected by x-ray films, and enzyme tags can be localized using colorimetric assays. The western blot c

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