Use this image of a generic cloning vector to answer questions 21-23. HindIII SphI PstI DNA sequencing SalI AccI HincII primer site XbaI 21 BamHI Multiple SmaI cloning site XmaI KpnI BanII Ampicillin lacZ SstI resistance gene EcoRI 23 gene (ampR) DNA sequencing primer site Origin of 22 replication (ori) 21) What is the purpose of a multiple cloning site? a. allows the vector to replicate independent of the host's genome b. contains known sequences to allow the vector to be sequenced c. the region in which a DNA fragment is cut and pasted d. a selectable marker to identify cells transformed by the vector 22) What is the purpose of the origin of replication? a. allows the vector to replicate independent of the host's genome b. contains known sequences to allow the vector to be sequenced c. the region in which a DNA fragment is cut and pasted d. a selectable marker to identify cells transformed by the vector 23) What is the purpose of the ampicillin resistance gene? a. allows the vector to replicate independent of the host's genome b. contains known sequences to allow the vector to be sequenced c. the region in which a DNA fragment is cut and pasted d. a selectable marker to identify cells transformed by the vector
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DNA Cloning The plasmid cloning vector pBR322 (see Fig. 9-3) is cleaved with the restriction endonuclease PstI. An isolated DNA fragment from a eukaryotic genome (also produced by PstI cleavage) is added to the prepared vector and ligated. The mixture of ligated DNAs is then used to transform bacteria, and plasmid-containing bacteria are selected by growth in the presence of tetracycline. (a) In addition to the desired recombinant plasmid, what other types of plasmids might be found among the transformed bacteria that are tetracycline-resistant? How can the types be distinguished? (b) The cloned DNA fragment is 1,000 bp long and has an EcoRI site 250 bp from one end. Three different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis, giving the patterns shown below. What does each pattern say about the cloned DNA? Note that in pBR322, the PstI and EcoRI restriction sites are about 750 bp apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted.
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Describe the process of molecular cloning. a. The foreign DNA and plasmid are cut with the same restriction enzyme and DNA is inserted within the lacZ gene, whose product metabolizes lactose. The foreign DNA and vector are allowed to anneal. The vector is transferred to a bacterial host that is ampicillin sensitive and those with a disrupted lacZ gene show inability to metabolize X-gal. b. The foreign DNA and plasmid are denatured using high heat, and DNA is inserted within the lacZ gene, whose product metabolizes glucose. The foreign DNA and vector are allowed to anneal. The vector is transferred to a bacterial host that is ampicillin sensitive and disrupted lacZ gene will metabolize X-gal c. The foreign DNA and plasmid are cut with the same restriction enzyme and DNA is inserted randomly in the plasmid. The foreign DNA and vector are allowed to anneal. The vector is transferred to a bacterial host that is ampicillin sensitive and the disrupted lacZ gene shows inability to synthesize X-gal. d. The foreign DNA and plasmid are cut with the same restriction enzyme and DNA is inserted within the lacZ gene, whose product metabolizes lactose. The foreign DNA and vector are allowed to anneal. The vector is transformed into a viral host that is ampicillin sensitive and the disrupted lacZ gene show inability to synthesize X-gal.
DNA Cloning The plasmid cloning vector $\mathrm{pBR} 322$ (see Fig. $9-3$ ) is cleaved with the restriction endonuclease $P$ stl. An isolated DNA fragment from a cukaryotic genome (also produced by PstI cleavage) is added to the prepared vector and ligated. The mixture of ligated DNAs is then used to transform bacteria, and plasmid-containing bacteria are sclected by growth in the presence of tetracycline. (a) In addition to the desired recombinant plasmid, what other types of plasmids might be found among the transformed bacteria that are tetracycline-resistant? How can the types be distinguished? (b) The cloned DNA fragment is 1,000 bp long and has an EcoRI site 250 bp from one cnd. Three different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis, giving the patterns shown below. What does each pattern say about the cloned DNA? Note that in $\mathrm{pBR} 322$, the Pst and EcoRI restriction sites are about $750 \mathrm{bp}$ apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted. FIGURE CANT COPY
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