Question

When working with your DNA with two enzymes each requiring different digest conditions, what is the correct step?

A. Perform the enzyme reactions sequentially, adding more DNA after the first reaction.
B. Perform enzyme reactions sequentially, purifying the DNA before using it in the second reaction.
C. Add more ligase to stabilize the digests between reactions.
D. Add more ligase to the buffer.

Answer 1

The correct step when working with DNA and two enzymes that require different digest conditions is to perform enzyme reactions sequentially, purifying the DNA before the second reaction to ensure the activities of different enzymes do not interfere with each other.

Step-by-step explanation:

When working with DNA and two enzymes each requiring different digest conditions, the correct step is to perform enzyme reactions sequentially, purifying the DNA before using it in the second reaction. This can be explained through several steps involved in DNA manipulation:

• The cloning vector and the amplified DNA are first digested with restriction enzymes.
• Subsequently, the cloning vector is purified using an agarose gel, an essential step for removing any contaminants that may interfere with further reactions.
• The purified cloning vector is then treated with phosphatase to prevent self-ligation.
• Finally, the cloning vector and the amplified DNA are ligated together using DNA ligase, which is crucial in forming the recombinant DNA by joining the DNA fragments covalently.
• The recombinant DNA is then transformed into host cells to propagate.

This sequential approach and purification steps ensure that each enzyme's activity does not interfere with the other and that only the desired DNA fragments are carried forward in the process.