You want to clone the linear DNA fragment illustrated below (1000 bp in size, Pst sticky...
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You want to clone the linear DNA fragment illustrated below (1000 bp in size, Pstī sticky ends, a Ndel site at position 200) into the PstI restriction site of the vector pBR322, which contains both ampicillin and tetracycline resistance genes. 3. EcoR (4350) Hindill (30) Nde! PstI Pstl BamHI (370) Psti 200 1000 (3000) Ampicilin Resistance Tetracycline Resistance PBR322 Xmal (840) (4.36 kb) Origin of Replication Pull (2060) a) List the antibiotics which you would include into the agar plates that would allow bacterial cells to grow that have been transformed with: i) No plasmid. ii) Non-recombinant PBR322. iii) Recombinant PBR322 with the DNA fragment cloned into the PstI site. b) When all DNA molecules have the same sticky ends, an insert can ligate into a vector in either orientation (non-directional cloning). Draw the two recombinant plasmids that could result from joining this fragment into the plasmid in two different insert orientations. Clearly indicate the vector DNA, the inserted fragment, and the restriction enzyme sites for PstI, Ndel and PvuII on each drawing. Predict the band sizes that will be generated by digestion of the non-recombinant PBR322 c) and either of the two recombinant with the enzymes listed below. PstI PvuII Ndel + PvulI Non-recombinant pBR322 Recombinant PBR322 (orientation 1) Recombinant pBR322 (orientation 2) (p You want to modify plasmid PBR322 from a cloning plasmid into an expression vector, to specifically allow bacterial expression of genes cloned into the PvuII site. How can this be accomplished? Which will be the two most important sequences to include in your PBR322 modifications? Explain. You want to clone the linear DNA fragment illustrated below (1000 bp in size, Pstī sticky ends, a Ndel site at position 200) into the PstI restriction site of the vector pBR322, which contains both ampicillin and tetracycline resistance genes. 3. EcoR (4350) Hindill (30) Nde! PstI Pstl BamHI (370) Psti 200 1000 (3000) Ampicilin Resistance Tetracycline Resistance PBR322 Xmal (840) (4.36 kb) Origin of Replication Pull (2060) a) List the antibiotics which you would include into the agar plates that would allow bacterial cells to grow that have been transformed with: i) No plasmid. ii) Non-recombinant PBR322. iii) Recombinant PBR322 with the DNA fragment cloned into the PstI site. b) When all DNA molecules have the same sticky ends, an insert can ligate into a vector in either orientation (non-directional cloning). Draw the two recombinant plasmids that could result from joining this fragment into the plasmid in two different insert orientations. Clearly indicate the vector DNA, the inserted fragment, and the restriction enzyme sites for PstI, Ndel and PvuII on each drawing. Predict the band sizes that will be generated by digestion of the non-recombinant PBR322 c) and either of the two recombinant with the enzymes listed below. PstI PvuII Ndel + PvulI Non-recombinant pBR322 Recombinant PBR322 (orientation 1) Recombinant pBR322 (orientation 2) (p You want to modify plasmid PBR322 from a cloning plasmid into an expression vector, to specifically allow bacterial expression of genes cloned into the PvuII site. How can this be accomplished? Which will be the two most important sequences to include in your PBR322 modifications? Explain.
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Answer Answer a i No plasmid To allow cells to grow without any plasmid the agar plates should have ... View the full answer
Related Book For
Brock Biology of Microorganisms
ISBN: 978-0321897398
14th edition
Authors: Michael Madigan, John Martinko, Kelly Bender, Daniel Buckley
Posted Date:
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