الم 3. You want to clone the linear DNA fragment illustrated below (1000 bp in size, Psti sticky ends, a Ndel site at po

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الم 3. You want to clone the linear DNA fragment illustrated below (1000 bp in size, Psti 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. Eco Psti Nde! Pasti Bam 3703 1 200 1000 Pati 3600 Tetracycline Resistance PBR322 (4.36 kb) mat 040 oor Replication 1 2060 a) b) List the antibiotics which you would include into the agar plates that would allow bacterial cells to grow that have been transformed with: 1) No plasmid. il) Non-recombinant PBR322. ill) Recombinant PBR322 with the DNA fragment cloned into the Psti site. 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 Pvull on each drawing. Predict the band sizes that will be generated by digestion of the non-recombinant PBR322 and either of the two recombinant with the enzymes listed below. c) Psti Pvull Ndel + PVull Non-recombinant PBR322 Recombinant PBR322 (orientation 1) Recombinant PBR322 (orientation 2) d) You want to modify plasmid PBR322 from a cloning plasmid into an expression vector, to specifically allow bacterial expression of genes cloned into the Pvull site. How can this be accomplished? Which will be the two most important sequences to include in your PBR322 modifications? Explain.