A virus with a circular double-stranded DNA chromosome contains approximately 10,000 bp. You want to begin characterizing this chromosome by making a map of the cleavage sites of three restriction endonucleases: EcoRI, Hind III, and BamHI. You digest the viral DNA under conditions that allow the endonuclease reactions to go to completion and then subject the digested DNA to electrophoresis on agarose to determine the lengths of the restriction fragments produced in each reaction. Based on the resulting data, draw a map of the viral chromosome indicating the relative positions of the cleavage sites for these restriction endonucleases: Endonuclease EcoRI HindIII BamHI EcoRI+HindIII EcoRI + BamHI HindIII+BamHI EcoRI + HindIII + BamHI Length of fragments (kb) 6.9,3.1 5.1, 4.4, 0.5 10.0 3.6, 3.3, 1.5, 1.1, 0.5 5.1, 3.1, 1.8 4.4, 3.3, 1.8, 0.5 3.3, 1.8, 1.5, 1.1, 0.5

A 2.0kb bacterial plasmid ‘BS1030’ is digested with the restriction endonuclease Sau3A; the plasmid map is depicted in the diagram below and the Sau3A (S) restriction sites are indicated. Which of the following DNA fragments do you expect to see on an agarose gel when you run Sau3A-digested plasmid ‘BS1030’ DNA?   a. 250 bp, 450 bp, 550 bp, 1.1 kb, 1.5 kb and 2.0 kb   b. 2.0kb   c. 250 bp, 400 bp, 450 bp, 500 bp and 550 bp   d. 100 bp, 200 bp, 250 bp, 400 bp, 500 bp and 550 bp

Assume that a plasmid is 4700 base pairs in length and has restriction sites for a given restriction enzyme at the following locations: 800, 1400, 2900, and 3600. List the fragments by size that are ! expected when the plasmid is fully digested the restriction enzyme.

Why would you NOT expect a restriction endonuclease to exist that would recognize the site AAGGAA?

Below is shown a plasmid map from a recombinant DNA experiment you have performed. You inserted your gene of interest (labeled gene on the map) into the lac Z reporter gene via a Hind III site. If you digested this recombinant DNA with HindIII what is the size of the DNA fragment(s) you would obtain on a DNA gel electrophoresis? The plasmid alone is 4000 bp. I have attached the image below.

Restriction endonucleases are bacterial enzymes that cleave duplex (double-stranded) DNA at specific nucleotide sequences. The mode of replication of the animal virus SV40 has been investigated by using restriction endonucleases that cleave SV40 DNA into a number of unique segments. Like most viruses, SV40 DNA is circular. The map positions of the 11 fragments produced by a pair of restriction endonucleases are shown on the next page. Immediately following a 5 or 10 minute pulse of radioactively labeled thymidine, labeled SV40 molecules that have completed replication during the pulse are isolated. These newly replicated DNA molecules are digested by the restriction endonucleases and the resulting fragments are analyzed for the relative amounts of pulse label they contain. The results are in the table below. Assume that at the time the label was added there was a random population of replicating SV40 DNA molecules in all possible stages of synthesis. From the information given below,…

Nucleosomes can be assembled onto defined DNA segments. When a particular 225-bp segment of human DNA was used to assemble nucleosomes and then incubated with micrococcal nuclease, which digests DNA that is not located within the nucleosome, uniform fragments 147  bp in length were generated. Subsequent digestion of these fragments with a restriction enzyme that cuts once within the original 225-bp sequence produced two well-defined bands at 37 bp and 110 bp. Why do you suppose two well-defined fragments were generated by restriction digestion, rather than a range of fragments of different sizes? How would you interpret this result?

Consider the ends of the DNA fragments shown below. They have been produced by digestion of a single sequence of DNA using a number of restriction endonucleases. 1. 5'A         3' 3'TTCGA5' 2. 5'G          3' 3'CAGCT5'     3. 5'AATTC3' 3'         G5 4. 5'TCGAC3' 3'         G5'     5. 5'GGG 3' 3'CCC 5'   Which of these ends are capable of annealing and being joined by DNA ligase?

Many of the gene products involved in DNA synthesis wereinitially defined by studying mutant E. coli strains that could notsynthesize DNA. Question: The dnaE gene encodes the a subunit of DNA polymeraseIII. What effect is expected from a mutation in this gene?How could the mutant strain be maintained?

Now that you’ve isolated the gene and made lots of copies, you need to insert the gene into something you can manipulate and move into your lab strain of E. coli.  You have access to the following vectors, either pBR322 or pUC19 (look them up here to see a map https://www.neb.com/products/dna-plasmids-and-substrates Please note the restriction sites in BOLD appear only once in the plasmid).  You may use either vector.  What is a vector? Which vector did you choose? Explain why you made that choice. What enzyme(s) will you use to place your fragment into the vector? Explain why you chose these enzymes. How will you move this construct into coli? What phenotype will tell you if the coli have taken up the plasmid? What phenotype will tell you if the coli have taken up the plasmid with the gasP gene?