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

dNTPs (deoxynucleotide triphosphates): The building blocks (A, T, C, G) required for DNA synthesis.

MgCl₂: A cofactor essential for DNA polymerase activity.

Buffer: Provides optimal pH and ionic conditions for the PCR reaction.

Stabilizers and enhancers: Improve the efficiency and specificity of the PCR reaction.

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

• Primer length: Longer primers generally require higher annealing temperatures.
• GC content: Primers with higher GC content require higher annealing temperatures due to stronger hydrogen bonding.
• Primer sequence: Specific sequences can influence the melting temperature (Tm) of the primer-template duplex.
• Salt concentration: Higher salt concentrations stabilize DNA duplexes, allowing for higher annealing temperatures.
• PCR buffer composition: The pH and ionic strength of the buffer can affect annealing efficiency.

1. There are two methods in this protocol that create linear fragments of DNA: PCR, and restriction enzyme digest. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other.

• PCR:
  • Protocol: Involves denaturation, annealing, and extension steps using primers and DNA polymerase.
  • Advantages: Can amplify specific regions of DNA, even from small amounts of template. No need for restriction sites.
  • When to use: When you need to amplify a specific region or introduce mutations, or when restriction sites are not available.
• Restriction Enzyme Digest:
  • Protocol: Involves cutting DNA at specific recognition sites using restriction enzymes.
  • Advantages: Simple and fast for linearizing plasmids or cutting out specific fragments.
  • When to use: When the DNA already contains appropriate restriction sites and you need to generate precise cuts.

Contrast:

• PCR is more versatile and can amplify DNA, while restriction digest is limited to cutting at specific sites.
• PCR requires knowledge of the DNA sequence for primer design, while restriction digest requires knowledge of restriction sites.