Name Restriction Digests II 1. On the plasmid below, you have ligated an insert
ID: 217859 • Letter: N
Question
Name Restriction Digests II 1. On the plasmid below, you have ligated an insert (shown below) into the polylinker at the BamHl site. You are going to send some of this final plasmid with the insert to several colleagues, who also, of course, want a map of the whole thing so they know exactly what they are getting. Do a mental digest of your plasmid to determine the prientation of the insert. Digest first with Pstl and then do a separate digestion with Hae3. What are the sines ofall the fragments would you expect to get with either enzyme? Now, figure out all the sizes you would get with either enzyme if the insert had gone in the other way around. And, which ones of those sizes are the distinquishing sizes that you are looking for? (Hint you can give approximate sizes in some cases, since I didn't give exact locations of polylinker sites. Nucleotide 6000/ 1 Pstl PstI 3550 1500 Hae3 3250 Polylinker EPASXH 3000 c s p a ba otacac Pst Hac3 Hae3 0 500 1500 1000 2000 These arrows indicate that the insert could have gone in either way.)Explanation / Answer
Forward orientation:
Pst I digestion: 1500 + 2450 + 3000 + 1000 = 7950 bp
HaeIII digestion: 2750 + 1250 + 500 + 3500 = 8000 bp
Reverse orientation:
Pst I digestion: 1500 + 2450 + 2000 + 2000 = 7950 bp
HaeIII digestion: 2750 + 750 + 500 + 4000 = 8000 bp
Bands that can be used to distinguish the orientation of the insert are highlighted in bold.
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