Homework Assignment (10 points) Question 1 A schematic of the vector p7012 is shown. The restriction enzymes listed cut only where indicated; they do not cut anywhere else in the vector or insert. a) A schematic of gene W is below. You want to clone all of gene W DNA into the p7012 vector. Give three different strategies that you could use to clone gene W into p7012, and obtain colonies that contain a recombinant plasmid. * Strategy 1 uses the restriction enzyme(s) ______Kpnl________ to cut the vector and restriction enzyme(s) ____Kpnl_________ to cut Gene W Strategy 1 uses the restriction enzyme(s) _EcoRL and Sall_ to cut the vector and restriction enzyme(s) __EcoRl and Xhol__ to cut Gene W * Strategy 1 uses the restriction enzyme(s) ___EcoRl and Kpnl___ to cut the vector and restriction enzyme(s) __EcoRl and Kpnl__ to cut Gene W Question 2 You purify a protein from a plant cell that can act as a potential appetite suppressant. Owing to its possible commercial application you decide to clone the gene, Gene A, which encodes this protein. You isolate this gene from the plant cell, clone it into a plasmid vector and amplify it in the bacterial cells. ) You decide to use the following plasmid to clone Gene A.

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To achieve this you digest both the genomic DNA and plasmid DNA using a restriction enzyme. 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. * Which restriction enzyme (Kpn I, Hind III, Sal I or Xho I) did you use to digest Gene A for insertion in to the plasmid? ould not use Sal 1 and Hind III since they cut within gene A. You would also not use Xho 1 since it cuts only on one side of gene A. You would use Kpn 1 since it will give you intact gene A with promoter and has only one recognition sitein the plasmid vector. * Which restriction enzyme (Kpn I, Hind III, Sal I or Xho I) did you use to digest the plasmid before insertion of Gene A?

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Briefly explain why. Kpn 1 as well, ensuring that the vector and insert have complementary ends for ligation. ) Youthen plate these transformed bacterial cells onto media that will allow you to distinguish between bacterial cells that obtained the plasmid and those that did not. Onto what type of growth medium will you plate your transformation mix? Explain your answer. You will plate the transformation mix in minimal media that contains ampicillin as the selection marker since the bacterial cells have an intact ampr gene. You will not use tetracycline as the selection marker since the tetr gene is disrupted in the recombinant plasmids that have the Gene A insert.

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