https://pages.htgaa.org/2026a/elsa-muleya/homework/week-06-hw-genetic-circuits-part-i/index.htmlAssignment: DNA Assembly 1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose? Phusion DNA Polymerase: This is the “engine.” It’s a highly thermostable enzyme that synthesizes new DNA strands. It’s “High-Fidelity” because it has $3’ \rightarrow 5’$ exonuclease activity (proofreading), making significantly fewer mistakes than standard Taq. dNTPs (Deoxynucleotide Triphosphates): These are the molecular building blocks (A, T, C, and G) used by the polymerase to construct the new DNA strand. Buffer (containing $Mg^{2+}$): Maintains the optimal pH for enzymatic activity and provides essential divalent cations. Magnesium ions act as a cofactor for the polymerase, helping it catalyze the phosphodiester bond. Stabilizers: Often includes detergents or proprietary chemicals to prevent the enzyme from denaturing or sticking to the tube walls during the high-heat cycles. 2. What are some factors that determine primer annealing temperature during PCR? Primer Length: Longer primers generally require higher temperatures to remain specific. GC Content: G-C pairs have three hydrogen bonds compared to the two in A-T pairs. Therefore, primers with higher GC content have higher melting temperatures ($T_m$). Salt Concentration: The concentration of monovalent cations (like $K^+$) in the buffer affects the stability of the DNA duplex. Primer Concentration: Higher concentrations can slightly shift the kinetics of annealing. Mismatches: If the primer isn’t a 100% match to the template, the $T_m$ will decrease. Note: The annealing temperature ($T_a$) is usually chosen to be $3-5^\circ\text{C}$ below the $T_m$ of the primers to balance specificity and yield.Hugoen