1. (8 points) Gene x is a mouse gene that codes for protein x. You are intereste
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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 EL.AY PUC 17 puCIgExplanation / Answer
The enzyme that can be used to isolate the gene X from mouse genome is bam H1. the same site is available on puc 17 o it can be used for cloning purposes.
the procedure to clone is as follows:
the gene is isolated from the genome using restriction digestion or PCR with Bam h1 at both ends.
plasmid puc 17 is digested with Bam H1 and ligatation reaction is setup between cut plasmid and gene X.
the resulted DNA is transformed into the cell.
the cells are allowed to grow on AMPICILLIN plate.
The cells that grows are either having the recombinant plasmid or just the plasmid.
grow each colony in a LB media and isolate plasmid.
run the gel of each isolation.
the plasmid that is having gene X incorporated would be larger in size and hence will run less distance on gel.
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