After digesting the DNA produced by PCR (generated using the primers mentioned in question 2 added for context below) and the pET-28a vector with restriction enzymes SacI and HindIII, what steps would be needed to obtain a pure culture of E. coli cells that contain the pET-28a-OXA-M290 plasmid (i.e., a culture which consists only of E. coli cells that contain this plasmid)? And, how could you confirm that the OXA-M290 gene was successfully cloned into the pET-28a vector without any mutations? [4 – 8 sentences suggested] Question 2 (Do not answer, just for context): The forward primer that will be designed is the primer that binds to the region of the OXA-M290 gene where the start codon is located, and the reverse primer is the primer that binds to the region of the gene where the stop codon is located. In order to clone the PCR product generated using these primers into pET-28a, extra nucleotides must be added to the 5' ends of the primers. These extra nucleotides will contain the recognition sites for the restriction enzymes that are used for cloning. When the DNA produced by PCR using these primers is treated with the restriction enzymes, this will generate sticky ends at the ends of the PCR product. If pET-28a is digested with the same restriction enzymes, the sticky ends of the PCR product will bind to the sticky ends on the digested pET-28a, allowing the two DNA molecules to be connected together by DNA ligase. Which primer (forward or reverse) should contain the SacI recognition site, and which primer (forward or reverse) should contain the HindIII cleavage site? Why? Hint: consider where the promoter is located in the pET-28a vector, relative to the multiple cloning site.
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After digesting the DNA produced by PCR (generated using the primers mentioned in question 2 added for context below) and the pET-28a vector with restriction enzymes SacI and HindIII, what steps would be needed to obtain a pure culture of E. coli cells that contain the pET-28a-OXA-M290 plasmid (i.e., a culture which consists only of E. coli cells that contain this plasmid)?
And, how could you confirm that the OXA-M290 gene was successfully cloned into the pET-28a vector without any mutations? [4 – 8 sentences suggested]
Question 2 (Do not answer, just for context):
The forward primer that will be designed is the primer that binds to the region of the OXA-M290 gene where the start codon is located, and the reverse primer is the primer that binds to the region of the gene where the stop codon is located.
In order to clone the PCR product generated using these primers into pET-28a, extra
Which primer (forward or reverse) should contain the SacI recognition site, and which primer (forward or reverse) should contain the HindIII cleavage site? Why?
Hint: consider where the promoter is located in the pET-28a vector, relative to the multiple cloning site.
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