Question

KpnI (255) lacz alpha gene ECORI (265) Psti (270) PUC origin Smal (3660) рвозобw 3973 bp EcoRI (875) Amp(R) Psti (1550) Kan(R) Ncol (1275) 8. You will be using the plasmid shown above as a vector for a DNA cloning experiment. You will create a library of genomic DNA fragments that will then be ligated into this plasmid. The plasmid carries two antibiotic resistance genes, Amp(R) and Kan(R), an origin of replication and a LacZ gene (Lac Z alpha). The recognition sequences for different restriction sites are also indicated. Based on the map of this plasmid, what enzyme would you choose for cutting the genomic DNA and the plasmid? Explain why you chose this enzyme as well as why you would not choose each of the other enzymes. 9. For the recombinant DNA constructed in question 9, how would you identify cells that (A) carry the plasmid and (B) whether or not the plasmid is recombinant.

Answer 1

8. We would use restriction enzymes that recognize specific 4 to 8 bp sequences known as restriction sites in the DNA or plasmid and cut them at this site.Other enzymes will not be able to cut at the specified sites.

9.

A.The cells that carry the plasmid would be able to grow on a media containing the antibiotic Amp and KanR as they would contain the gene for the antibiotics that would render them antibiotic resistant.

B.The plasmid is recombinant or not can be identified via blue white colonies.Any colony that will contain the plasmid with the β-galactosidase gene will turn blue due to the activity of the β-galactosidase activity.