. You are working with a genomic DNA library. This library contains human genomi
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. You are working with a genomic DNA library. This library contains human genomic DNA cloned into the pcDNA plasmid. You want to insert your genomic DNA containing pcDNA recombinant plasmids into bacteria, then purify the plamids and determine the DNA sequence. But you don ?t make sure that each plamid enters a single bacterium. (You use too many plasimids per bacteria). You then clone a single bacterium with recombinant DNA. purify plasmid DNA and perform chain - termination sequencing reaction. The result is shown below. When you look at this sequencing result you conclude that you have two different plasmids in this bacterial clone but not 3 or 4. You repeat the above experiment by making sure that one plasmid enters one bacterium and when you repeat your sequencing reaction you realize that this is indeed the case: you have one recombinant plasmid in one bacterium, This time. you want to determine the size of the genomic DNA inserted into the plasmid by restriction digestion because you don?t know the size of the genomic DNA fragment in the plasmid. pcDNA contains an EcoRl site. You also know that when preparing the recombinant plasmid. you had digested pcDNA with EcoRI before ligating it with the genomic DNA. which was also digested with EcoRl. To determine the size of the insert, after you purify your plasmid. you design the following gel electrophoresis experiment: (figure on the next page) Lane 1: molecular weight markers (k = 1000 base pairs) Lane 2: pcDNA (not containing a genomic DNA insert), digested with EcoRl Lane 3: pcDNA (containing your genomic DNA insert), digested with EcoRI Lane 4. pcDNA (containing your genomic DNA insert, not digested with EcoRI Based on the results shown on the left. you reach the two conclusions described below. Please explain why you reach these conclusions. Explain your logic briefly, a. Conclusion 1: The 2enomic DNA insert contains a EcoRl recognition site close to the middle of the DNA insert. b. Conclusion 2: The length of the genomic DNA fragment in pcDlA is about 5k (5000 base pairs).Explanation / Answer
a. The pcDNA in lane 2 is very close to genomic DNA in lane 3 shows the presence of genomic DNA fragment.
b. Relative mobility and bond intencity when compared with reference to ladder gives size of the DNA fragment.
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