Assume that you are trying to insert a gene into a plasmid. Someone gives you a preparation of genomic DNA that has been 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
A) insert the fragments cut with restriction enzyme X directly into the plasmid without cutting the plasmid.
B) cut the plasmid with restriction enzyme X and insert the fragments cut with restriction enzyme Y into the plasmid.
C) cut the DNA again with restriction enzyme Y and insert these 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 DNA fragments cut with restriction enzyme X.
E) cut the plasmid with restriction enzyme X and then insert the gene into the plasmid.

Question

Quizplus · Accepted Answer

Since the gene to be inserted has sites for restriction enzyme Y and the plasmid has a single site for Y but not for X, the correct strategy is to cut the DNA (which contains the gene) with restriction enzyme Y and then insert these fragments into the plasmid that has been cut with the same enzyme (Y). This ensures compatibility between the ends of the gene and the plasmid, facilitating successful ligation.

Assume That You Are Trying to Insert a Gene into a Plasmid