https://pages.htgaa.org/2026a/juan-larrea/homework/week-6-genetic-circuits-part-i---assembly-technologies/index.htmlMolecular Biology: PCR, Cloning & Transformation 1. Phusion High-Fidelity PCR Master Mix Components Phusion Hot Start II DNA Polymerase — synthesizes new DNA strands; has 3’→5’ exonuclease (proofreading) activity to correct misincorporated bases, giving very high fidelity. dNTPs (dATP, dCTP, dGTP, dTTP) — deoxynucleotide triphosphates; the building blocks incorporated into the growing DNA strand. MgCl₂ (magnesium chloride) — essential cofactor; Mg²⁺ ions stabilize the enzyme-DNA-dNTP complex and are required for catalytic activity. Optimized reaction buffer — maintains correct pH and ionic environment for efficient polymerase activity and primer annealing. Hot-start antibody/aptamer — inhibits polymerase at room temperature to prevent non-specific amplification; releases the enzyme once the initial high-temperature denaturation step is reached. 2. Factors Determining Primer Annealing Temperature GC content — G-C pairs have 3 hydrogen bonds vs. 2 for A-T; higher GC → higher Tm → higher annealing temperature. Primer length — longer primers have higher Tm due to more base-pair contributions to stability. Self-complementarity — hairpins or primer dimers reduce effective annealing temperature. Salt/ion concentration — higher Mg²⁺ or monovalent cations stabilize the duplex, raising Tm. Additives (formamide, DMSO) — destabilize base pairing, lowering effective Tm; useful for GC-rich regions. Mismatches — imperfect complementarity (e.g., mutagenic primers) requires lower annealing temperature. 💡 Rule of thumb: set annealing temperature ~5°C below the calculated Tm of the primer pair.Hugoen