A circular bacterial plasmid containing a gene for tetracycline resistance was c
ID: 22783 • Letter: A
Question
A circular bacterial plasmid containing a gene for tetracycline resistance was cut with restriction enzyme BglII. Electrophoresis showed one band of 14 kb.a. How many BglII restriction sites are there in this plasmid?
The plasmid was cut with EcoRV and electrophoresis produced two bands, one of 2.5 kb and the other 11.5 kb.
b. How many EcoRV restriction sites are there in this plasmid?
Digestion with both enzymes together resulted in three bands of 2.5, 5.5, and 6 kb.
c. What can be deduced from this result?
Plasmid DNA cut with BglII was mixed and ligated with donor DNA fragments, also cut with BglII, to make recombinant DNA molecules. All recombinant clones proved to be tetracycline sensitive.
d. Give the most likely reason why the recombinant molecules lost their tetracycline resistance capabilities.
One recombinant clone was cut with BglII, and fragments of 4 and 14 kb were observed.
e. What is the size of the fragment that was ligated into the plasmid?
The same clone was treated with EcoRV and fragments of 2.5, 7, and 8.5 were observed.
f. Explain these results by showing a BglII and EcoRV restriction map of the recombinant DNA. (the top
Explanation / Answer
You have a purified DNA molecule, and you wish to map restriction-enzyme sites along its length. After digestion with EcoRI, you obtain four fragments: 1, 2, 3, and 4. After digestion of each of these fragments with HindII, you find that fragment 3 yields two subfragments (31 and 32) and that fragment 2 yields three (21, 22, and 23). After digestion of the entire DNA molecule with HindII, you recover four pieces: A, B, C, and D. When these pieces are treated with EcoRI, piece D yields fragments 1 and 31, A yields 32 and 21, and B yields 23 and 4. The C piece is identical with 22. Draw a restriction map of this DNA. . After Drosophila DNA has been treated with a restriction enzyme, the fragments are attached to plasmids and selected as clones in E. coli. By using this “shotgun” technique, David Hogness has recovered every DNA sequence of Drosophila in a library. You have isolated and cloned a segment of DNA that is known to be a unique sequence in the genome. It maps near the tip of the X chromosome and is about 10 kb in length. You label the 5' ends with 32P and cleave the molecule with EcoRI. You obtain two fragments: one is 8.5 kb long; the other is 1.5 kb. You separate the 8.5-kb fragment into two fractions, partly digesting one with HaeIII and the other with HindII.
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