A hybrid recombinant plasmid is constructed from pBR325 by the insertion of a fragment of DNA at the BamHI restriction site. How could you most easily determine the size of the inserted fragment?

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Nestor RutherfordLv2

3 Aug 2019

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Q1. A researcher tests two different restriction enzymeson pUC18, performing the pUC18 digests then running them in anagarose gel against linear standards. The first enzyme produced theexpected single linear band of ~2.7 kb, but the second digestproduced a single band of only about 0.85 kb. (a) Explain thissecond digest result. (b) What telltale clue in the gel profilewould lead you to the correct answer for part (a)?

Q2. A DNA fragment, obtained from a partial SmaI digestof a longer fragment, is ligated into the unique SmaI site of thecloning vector, pUC18. A subsequent and complete SmaI digest of newrecombinant plasmid yielded a total of four SmaI fragments. Howmany SmaI sites are there in this recombinant plasmid? How manySmaI sites were in the original partially digested fragment(careful!)?

Clue for Q2: Therecombinant plasmid has

coralminnow616

Clue for Q2: Therecombinant plasmid has

lemonrhinoceros823

1. Below is a map of a plasmid called pBR322:

(Cannot figure out how to post the picture)

Suppose that the pBR322 plasmid is cleaved with the restriction enzyme PstI. Suppose a PstI-digested segment of mouse DNA is then ligated into the PstI site in pBR322. This recombinant DNA molecule is then used to transform E. coli cells. a. Which antibiotic should be added to the medium to select for cells that have incorporated a recombinant plasmid? b. How can you explain the presence of bacterial colonies that are resistant to both antibiotics? c. You decide to digest your recombinant DNA molecule with BamHI and EcoRI and then analyze the digested recombinant DNA molecule on an agarose gel. How many bands will you see on the gel? d. You decide to digest your recombinant DNA molecule with BamHI and PstI and then analyze the digested recombinant DNA molecule on an agarose gel. How many bands will you see on the gel? ?a. Which antibiotic should be added to the medium to select for cells that have incorporated a recombinant plasmid?

b. How can you explain the presence of bacterial colonies that are resistant to both antibiotics?

c. You decide to digest your recombinant DNA molecule with BamHI and EcoRI and then analyze the digested recombinant DNA molecule on an agarose gel. How many bands will you see on the gel?

d. You decide to digest your recombinant DNA molecule with BamHI and PstI and then analyze the digested recombinant DNA molecule on an agarose gel. How many bands will you see on the gel?