A possible forward primer to be designed is a 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? [3 – 5 sentences suggested] Hint: consider where the promoter is located in the pET-28a vector, relative to the multiple cloning site.

Human Anatomy & Physiology (11th Edition)
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A possible forward primer to be designed is a 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? [3 – 5 sentences suggested]

Hint: consider where the promoter is located in the pET-28a vector, relative to the multiple cloning site.

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