https://pages.htgaa.org/2026a/devorah-wertheimer/homework/week-06-hw-genetic-circuits-part-i/index.htmlHW Questions What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? Phusion DNA Polymease, which synthesized new DNA by adding new nucleotides to the template DNA during PCR dNTPs, which are the nucleotide building blocks (dATP, dGTP, dCTP, and dTTP) reaction buffer, which acts as a chemical stabilizer that maintains the ideal pH and salt balance so the enzyme stays active and can accurately build new DNA strands. What are some factors that determine primer annealing temperature during PCR? Melting temperature of the primer, which is the temperature at which half of the DNA complex dissociates Primer length, since longer primers usually require higher annealing temperatures GC content, since higher GC content typically increases the primer melting temperature Salt concentration, since higher salt concentrations can stabilize the DNA and thus may require higher annealing temperatures There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other. Feature PCR (Polymerase Chain Reaction) Restriction Enzyme Digest Mechanism Enzymatic Synthesis: Building new strands from primers. Enzymatic Cleavage: Cutting phosphodiester bonds at specific sites. Protocol Thermal Cycling: Repeated steps of denaturation (95°C), annealing (55-65°C), and extension (72°C). Isothermal Incubation: DNA and enzymes are mixed in a buffer and held at a constant temp (usually 37°C). Reagents DNA template, Primers, dNTPs, Taq Polymerase, MgCl2, Buffer. DNA template, Restriction Enzymes, specific BSA/Salt Buffer, Water. Pros High sensitivity; amplifies DNA; creates specific fragments without needing existing cut sites. Simple setup; highly reproducible; great for verifying known sequences or circular DNA. Cons Prone to contamination; requires known flanking sequences; potential for polymerase errors. Does not amplify DNA; limited by the location of natural recognition sites. When to Use When you have minimal DNA, need a custom fragment, or want to add “tails” for cloning. When linearizing plasmids, performing diagnostic checks, or subcloning existing inserts. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning? Both the PCR and digested fragments must share identical overlapping terminal sequences (15–40 bp) with their neighboring fragments to allow for seamless homology-directed assembly. How does the plasmid DNA enter the E. coli cells during transformation? Membrane pores open due to a thermal pressure imbalance during the heat shock, allowing the plasmid DNA (which has been neutralized by calcium ions) to be pulled into the cell. Describe another assembly method in detail (such as Golden Gate Assembly) Golden Gate Assembly is a highly efficient “one-pot” cloning method that allows you to join multiple DNA fragments together simultaneously using Type IIS restriction enzymes and T4 DNA ligase. Unlike standard enzymes, Type IIS enzymes like BsaI bind to a specific recognition sequence but cut the DNA several nucleotides away, creating custom 4-base overhangs. By strategically designing these overhangs to be complementary, you can ensure that multiple fragments assemble in a specific, directional order. During the reaction, you cycle the temperature to repeatedly cut and ligate the DNA until the fragments are perfectly joined. A key advantage is that the enzyme’s recognition sites are positioned to be “cut off” and removed during the process, meaning the final product cannot be re-cut. This makes the reaction irreversible and drives the assembly toward the final, seamless circular plasmid. Because of this precision, Golden Gate is the gold standard for modular cloning and building complex multi-gene constructs. Simulating Golden Gate using AddGene’s toolHugoen