Arrange the steps involved in gene cloning using bacteria in the correct order.

Question

Arrange the steps involved in gene cloning using bacteria in the correct order.

Purify recombinant plasmid DNA from the bacterial colonies for further use, or purify the protein encoded by the cloned gene.

Treat the plasmid and bacteria mixture with heat and calcium so that the cell membrane allows the plasmid to pass into the cell, and then allow the bacteria to grow in solid medium (food) containing an antibiotic.

Use a restriction enzyme (let's call it restriction enzyme A) to cut the gene that you want to clone out of its chromosome.

Cut the plasmid with a restriction enzyme (let's call it restriction enzyme

A), so that the DNA loop opens up into a linear chain.

Mix the DNA (already cut with the restriction enzyme) with the linear plasmid. This is called a recombinant plasmid, or recombinant DNA.

Mix the recombinant plasmid with E. coli bacterial cells.

Explanation / Answer

1) Use a restriction enzyme (let's call it restriction enzyme A) to cut the gene that you want to clone out of its chromosome.

2) Cut the plasmid with a restriction enzyme (let's call it restriction enzyme A), so that the DNA loop opens up into a linear chain.

3) Mix the DNA (already cut with the restriction enzyme) with the linear plasmid. This is called a recombinant plasmid, or recombinant DNA.

4) Mix the recombinant plasmid with E. coli bacterial cells.

5) Treat the plasmid and bacteria mixture with heat and calcium so that the cell membrane allows the plasmid to pass into the cell, and then allow the bacteria to grow in solid medium (food) containing an antibiotic.

6) Purify recombinant plasmid DNA from the bacterial colonies for further use, or purify the protein encoded by the cloned gene.

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