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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?

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Jarrod Robel
Jarrod RobelLv2
28 Sep 2019
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Related questions

A kanamycin-resistant, ampicillin-resistance plasmid (3 kb) is cleaved with the PstI enzyme, which cuts within the ampicllin resistance gene. The digested plasmid is ligated to a 5-kb PstI DNA fragment from Drosophila (fruit fly). The DNA mixture was then transformed into E.coli host cells.

(i). Which antibiotic would you put into the medium (nutrient agar plate) to select for cells that contain the plasmid?

(ii). Explain step by step, how you would select for cells containing the recombinant Drosophila DNA.

(iii) If different colonies from the plate in part (ii) are cultured in nutrient broth and DNA was isolated from each of the bacterial cultures, what restriction patterns would you expect to see following PstI digestion and agarose gel electrophoresis?

An ampicillin-resistant, tetracycline-resistant plasmid iscleaved with EcoRI, which cuts within the tetracycline gene. Thecut plasmid is ligated with EcoRI-digested Drosophila DNA toprepare a genomic library. This DNA is then used to transform E.coli K12. A) Under what conditions should you grow bacteria inorder to select for transformed cells (those that have taken up aplasmid)? B) How can you identify bacteria carrying a plasmid thatcontains a fragment of the Drosophila DNA? C) How can you explainthe presence of colonies that are resistant to bothantibiotics?

A plasmid that carries two antibiotic resistance genes, to ampicillin and tetracycline, respectively, has one restriction site for EcoRI located within the tetracycline resistance gene. Eukaryotic DNA containing a gene of interest (g.o.i.) has been digested with diffferent restriction enzymes, none of which cut within the gene of interest sequence). The resultant fragments are added to plasmid DNA that has been digested with EcoR1 only; the mixture was then incubated with DNA ligase. Resulting plasmids (recombinant and otherwise) are added to competent bacteria lacking resistance genes and growing in nutrient broth. The bacteria are then plated and allowed to grow overnight producing isolated colonies. Samples of these colonies are then grown in four different media: nutrient broth plus ampicillin; nutrient broth plus tetracycline; nutrient broth plus ampicillin and tetracycline; and nutrient broth containing no antibiotics.

1. The bacteria containing the plasmid with the gene of interest successfully insertedin would grow in

A. Tthe nutrient broth only.

B. The nutrient broth, the ampicillin broth, and the tetracycline broth.

C. Tthe nutrient broth and the tetracycline broth only.

D. The ampicillin broth and the nutrient broth.E.Tthe ampicillin and tetracycline broth only.

2. Bacteria transformed with the plasmid alone (not containing any eukaryotic DNA), would grow

A. Only in the broth containing both antibiotics.

B. In the nutrient broth plus ampicillin, but not in the broth containing tetracycline.

C. In the broth containing tetracycline, but not in the broth containing ampicillin.

D. Only in the broth that contained no antibiotics.

E. In all four types of broth.

4. Bacteria that did not take up any plasmids at all would grow in which media?

A. The tetracycline and ampicillin broth only

B. The nutrient broth and the tetracycline broth only

C. The nutrient broth and the ampicillin broth only

D. The nutrient broth only

E. All four broths

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