1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

• Phusion DNA Polymerase: High-fidelity enzyme for accurate DNA amplification.
• dNTPs: Building blocks for new DNA strand synthesis.
• MgCl₂: Cofactor for polymerase activity.
• Buffer: Maintains pH and ionic strength for optimal enzyme function.
• Stabilizers: Enhance enzyme performance and stability during thermal cycling.

2. What are some factors that determine primer annealing temperature during PCR?

• GC content: Higher GC = higher Tₐ due to stronger hydrogen bonding.
• Primer length: Longer primers usually require higher Tₐ.
• Sequence mismatches: Decrease Tₐ.
• Salt concentration: Higher salt stabilizes DNA duplex, increasing Tₐ.
• Secondary structures: Hairpins or dimers can reduce primer efficiency.

3. Compare and contrast PCR and restriction digest for creating linear DNA fragments.

• PCR: Amplifies DNA using primers and polymerase; customizable and precise, especially for non-restriction site-based fragments.
• Restriction Digest: Uses enzymes to cut at specific sequences; faster and reliable if suitable sites exist.