https://pages.htgaa.org/2026a/yutong-wu/homework/week-06-hw-genetic-circuits-part-i/index.htmlPart 1. Questions 1. Phusion High-Fidelity PCR Master Mix Components Phusion DNA Polymerase — high-fidelity polymerase with 3′→5′ proofreading exonuclease activity; ~50× lower error rate than Taq dNTPs — nucleotide building blocks (dATP, dCTP, dGTP, dTTP) incorporated during strand synthesis HF Buffer + Mg²⁺ — provides optimal pH and ionic conditions; Mg²⁺ is an essential cofactor for polymerase activity Stabilizers — maintain enzyme activity during storage and reaction setup 2. Factors That Determine Primer Annealing Temperature GC content — G·C pairs have 3 H-bonds vs. 2 for A·T, raising T_m Primer length — longer primers = higher T_m Salt/Mg²⁺ concentration — stabilizes duplexes, increases T_m Primer secondary structure — hairpins or self-dimers reduce effective T_m Polymerase used — Phusion tolerates higher T_a than Taq; use NEB Tm Calculator for Phusion Rule of thumb: T_a ≈ T_m of the lower-melting primer (for Phusion)Hugoen