Kpn1 You are trying to clone Gene A into this 3kb plasmid using restriction enzymes. You then ligate the Gene A DNA into the cut plasmids. Finally, you transform the E. coli bacterial cells with the ligation mix (the recombinant plasmids). Note: The recognition sites for Kpn 1 and Sal1 on plasmid are 1 kb apart. Hindill 1 kb 3kb Sal1 Xho1 Bacterial ORI Xho1 Kont promoter Sal1 0.5kb Gene A 1.5kb Hindill Kpn1 Hindill a. Which restriction enzyme (Kpn 1, Hind III, Sal I or Xho 1) did you use to digest Gene A for insertion in to the plasmid? b. Which restriction enzyme (Kpn 1, Hind III, Sal I or Xho I) did you use to digest the plasmid before insertion of Gene A? Briefly explain why. c. Onto what type of growth medium will you plate your transformation mix? Explain your answer and include which antibiotic you would use. d. How can you tell which of the colonies isolated in c have an insert? e. You pick colonies from you transformation plate, extract DNA and cut with Kpnl. Draw the pattern you would get for plasmids that do not contain an insert, and those who do contain an insert. f. You want to know if your insert went in with the promoter on the left (which would allow for expression) or if it went on the right (backwards, no expression). Your advisor tells you to cut it with Sal I and figure it out. This is the pattern you observe. Which of these represents no insert? Which these represents direct insert? Which these represents backwards insert. 5 kb 3.5 kb 3 kb 2.5 kb 2 kb 1kb