Briefly describe the series of enzymatic treatments | Homework Help

You want to make an antibody against a C. elegans nematode protein that the DNA shown in Figure A encodes. To do this, you will clone the coding sequence into a bacterial expression plasmid, overexpress the protein, purify it, and inject it into a mouse to stimulate the production of antibodies against the protein.

A.         You use polymerase chain reaction (PCR) amplification to amplify the DNA corresponding to the 1800 base pairs of coding sequence; this coding sequence lies between the two flanking sequence shown in Figure A. You will then insert the PCR product into the BamHI site of the expression plasmid shown in Figure B. What are the sequences of the two oligonucleotide primers that will allow you to amplify the DNA by PCR and insert it into the BamHI site? (Remember to indicate the 5′ and 3′ ends of the primers.)

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B.         Briefly describe the series of enzymatic treatments you will use to create the expression plasmid before transferring it into bacterial cells.

C.         Several weeks after you inject a mouse with your purified protein, you sample its blood serum, which contains antibodies. You know that the mRNA corresponding to your protein is found in gonad cells but not in gut cells. To test whether the mouse has made antibodies against your protein, you isolate protein from gonad cells and from gut cells and perform a Western blot (also known as an immunoblot) with the mouse serum and a fluorescent second antibody that binds mouse antibodies. Your result is shown in Figure Did the mouse make antibodies against your protein? Does the serum specifically recognize only one nematode protein? (Illustrate your written answers by labeling the blot.)

 

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