Assume that you are trying to insert a gene into a plasmid and someone gives you a preparation of DNA cut with restriction enzyme X. The gene you wish to insert has sites on both ends for cutting by restriction enzyme Y. You have a plasmid with a single site for Y, but not for X. Your strategy should be to

Options

A. Cut the plasmid with restriction enzyme X and inert the fragments cut with Y into the plasmid
B. Cut the plasmid with restriction enzyme X and insert the gene into the plasmid
C. Cut the plasmid twice with restriction enzyme Y and ligate the two fragments into the plasmid cut with the same enzyme
D. Cut the plasmid twice with restriction enzyme Y and ligate the two fragments onto the ends of the human DNA fragments cut with restriction enzyme X

Answer

To solve the problem of inserting a gene into a plasmid using restriction enzymes, we can follow these steps:

### Step-by-Step Solution:

1. **Understand the Components**:
- You have a gene of interest that has restriction sites for enzyme Y at both ends.
- You have a plasmid that has a single site for enzyme Y but does not have a site for enzyme X.
- You also have DNA that has been cut with restriction enzyme X.

2. **Identify the Cutting Enzymes**:
- Since the plasmid has a site for enzyme Y, we will use this enzyme to cut the plasmid.
- The gene of interest can be inserted into the plasmid after both are cut with the same restriction enzyme.

3. **Cut the Plasmid with Restriction Enzyme Y**:
- Use restriction enzyme Y to cut the plasmid at its single site. This will create sticky or blunt ends depending on the type of cut made by enzyme Y.

4. **Prepare the Gene of Interest**:
- Ensure that the gene of interest is also cut with restriction enzyme Y. This will create compatible ends that can be ligated to the plasmid.

5. **Ligate the Gene into the Plasmid**:
- Mix the cut plasmid and the cut gene of interest together in the presence of DNA ligase. The ligase will join the compatible ends of the plasmid and the gene, forming recombinant DNA.

6. **Transform the Recombinant DNA into Host Cells**:
- Once the recombinant DNA is formed, it can be introduced into host cells (like bacteria) through a process called transformation, allowing the gene of interest to be expressed.

### Final Strategy:
The correct strategy is to **cut the plasmid twice with restriction enzyme Y and ligate the gene of interest into the plasmid cut with the same enzyme**.

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