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Then a polylinker was designed to be first inserted

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Then a polylinker was designed to be first inserted into the SwaI site, after which the 5’ arm will be subsequently cloned into the ploxPFlpneo plasmid utilizing one of the restriction sites in the “SwaI polylinker.” The SwaI polylinker sequence is shown below in capital letters: agacgcATTTAAATTCGAATTCCTCAGCAGATCTGGGCCCATTTAAATagacgc In the Linker: BbvC I = CC*TCAGC Bgl II = A*GATCT BstB I = TT*CGAA EcoR I = G*AATTC Apa I = GGGCC*C Swa I = ATTT*AAAT Outside the Linker: Swa I = ATTT*AAAT BamH I = G*GATCC Apa I = GGGCC*C Xho I = C*TCGAG * denotes where the restriction site is cut by the restriction enzyme.
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ploxPFlpneo plasmid schematic
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Questions: 1. What are some potential reasons as to why the 5’ arm was so difficult to clone into the SwaI restriction site (before the SwaI polylinker was introduced)? SwaI is a “blunt cut” restriction enzyme. Blunt cut restriction enzymes are less efficient than “sticky end” restriction enzymes. As such, larger DNA fragments are difficult to ligate in between SwaI and more ligase is required. 2. What is the most likely reason why the 5’ arm must be cloned into this plasmid before the 3’ arm? There is a SwaI restriction site within the 3’ arm; conversely, there is no XhoI site in the 5’ arm.
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