1. (8 points) Gene x is a mouse gene that codes for protein x. You are intereste

Question

1. (8 points) Gene x is a mouse gene that codes for protein x. You are interested in studying the function of protein x. To do so, you will first need to clone gene x. Below is the sequence of gene x as it is found in mouse genomic DNA. The gene is flanked by several restriction enzyme recognition sites, as indicated. There are two plasmids that you can chose from for your cloning project, shown below. Each plasmid has several restriction enzyme recognition sites, as indicated. pUC17 has the amp" gene (codes for a protein that confers ampicillin resistance). pUC18 has the tet gene (codes for a protein that confers tetracycline resistance). To work with, you also have E. coli cells and all of the enzymes and antibiotics that you could possibly need. Fully describe the approach that you would use to clone gene x, including the enzymes, plasmids, and antibiotics that you would use at each step. Be specific. Use the steps of molecular cloning that we discussed in class as a guide. (Note: you won't be using blue/white screening for your selection, just talk about what antibiotic you would use for selection.) Ava T PUC 17 puCIg

Explanation / Answer

1st approach: Using a single restriction enzyme

We can use BamHI to cleave the genomic sequence and pUC17 vector: Amp selection

We can use HindIII to cleave the genomic sequence and pUC18 vector: TetR selection

2nd approach: Using two restriction enzymes

We can use AvaI+BamHI or BamHI+EcoRV combination to cleave the genomic sequence and pUC17 vector: Amp selection

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