Plasmid Practice Problems Key

Run off the gel and be lost its not the optimal

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run off the gel and be lost), it’s not the optimal method for determining the presence of the SwaI polylinker. Digestion with ApaI will yield 2 bands (at about 3.0Kb and 2.2Kb). Both these bands are easily resolved on agarose gel, thus an ApaI digestion would be an appropriate test.
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6. Diagram what you would see on an agarose gel if you digested this plasmid with ApaI. Now diagram what you would see if you digested this plasmid at any other restriction site. Assume that all fragments (Exon 4, 5’ arm, and 3’ arm) have all been inserted and round to the nearest tenth of a Kb. Total size of plasmid = 4.8 + 3.1 + 3.0 + 0.38 + 0.05 = 11.3Kb ApaI digestion yields: 2 bands at about 6.1Kb and 5.2Kb All other restriction sites yields: 1 band at about 11.3Kb 7. Locate and diagram all the restriction sites in the SwaI polylinker. What do you notice? Swa I EcoR I Bgl II Swa I agacgcATTTAAATTCGAATTCCTCAGCAGATCTGGGCCCATTTAAATagacgc BstB I BbvC I Apa I You’ll notice that many of the restriction sites overlap, particularly at their ends. This helps to conserve nucleotides when synthesizing an oligonucleotide. Shorter oligonucleotides are easier to synthesize (as well as cheaper when ordering them from a company). Due to the overlap, one cannot do a digestion with any restriction enzymes with overlapping nucleotides. In this case, a “serial” digestion must be performed – where the sequence is digested in one restriction enzyme before being digested in the next.
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