https://pages.htgaa.org/2026a/anastasia-ntavou/homework/week-6/index.htmlWeek 6 HW: Gibson Assembly Questions 1. Phusion High-Fidelity PCR Master Mix Components [web:1266] Component Purpose Phusion DNA Polymerase High fidelity (52x Taq), fast extension dNTPs DNA building blocks MgCl₂ Polymerase cofactor (NH₄)₂SO₄ Stabilizes polymerase Betaine GC-rich templates DMSO Reduces secondary structure 2. Primer Annealing Temperature Factors [web:1267] Primer Tm (5°C below lowest Tm) Primer length (>20nt: +3°C above Tm) GC content (higher GC = higher Tm) Salt concentration (50mM default) Primer concentration (200-1000nM) 3. PCR vs Restriction Digest [web:1268] Feature PCR Restriction Digest Linear fragments Primers define ends Restriction sites Protocol 30 cycles (denature/anneal/extend) 1-2h 37°C digestion Advantages Scarless, any sequence Fast, cheap Gibson use Overlap primers (20-40bp) Compatible overhangs 4. Gibson Cloning Requirements [web:1269] 20-40bp overlaps between fragments No restriction sites in overlap regions High quality PCR (Phusion fidelity) Linearized vector (PCR or digest) Exonuclease chews back → Anneal → LigateHugoen