Question

Which of the following treatments can reduce the frequency of re-ligation of plasmid vector?

I Use a high insert to vector ratio in ligation
II. Remove the 5' phosphates of the vector by phosphatase
III. Digest the vector with two different restriction enzymes
IV. Purify the vector and insert before ligation
A. II and III
B. I, II and III
C. III and IV
D. I and II
E. All of the above

Answer 1

To reduce plasmid vector re-ligation, treatments II (removing the 5' phosphates of the vector by phosphatase) and III (digesting the vector with two different restriction enzymes) should be used.

Step-by-step explanation:

To reduce the frequency of re-ligation of a plasmid vector, the treatments II (removing the 5' phosphates of the vector by phosphatase) and III (digesting the vector with two different restriction enzymes) should be used. Removing the 5' phosphates prevents the vector from religating without the amplified DNA, while digestion with two different restriction enzymes increases the chances of getting the desired insertion. Therefore, the correct answer is A. II and III.