A 9kb fragment of human DNA that has EcoRI sticky ends is cloned into the plasmid PBR007 at its single EcoRI restriction site. The resulting recombinant plasmid is digested with EcoRI and then, separately, with BamHI. In addition, a third digest is performed, in which EcoRI and BamHI are used together. The resulting fragments are run out on a gel. Below are the diagrams of the gel patterns from each digest:
EcoRI 10kb 9kb
BamHI 8kb 6kb 5kb
EcoRI + BamHI 7kb 5kb 4kb 2kb 1kb
A. How many kbs is the PBR007 plasmid without the insert?
B. How many EcoRI and BamHI sites are present in the newly formed recombinant plasmid?
C. Draw a restriction map of the recombinant plasmid. In your map, distinguish between the human DNA fragment and the plasmid. Indicate the restriction sites and the distances between the restriction sites.
D. If Southern blot analysis was performed on the double digest using the original 9kb insert as a probe, which bands would this probe hybridize to?
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