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

“Phusion DNA Polymerase, deoxynucleotides and reaction buffer that has been optimized and includes MgCl2. All that is required is the addition of template, primers and water.”

DNA Polymerase: copies DNA strands and revises incorrect base pairs

Deoxynucleotides: dNTPs include the A, T, G, and C base pairs in the molecule - this is the source for adding base pairs during PCR

Reaction buffer with MgCl2: helps create an optimal environment for DNA polymerase because magnesium helps it bind to the DNA template strand

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

The melting temperature of the primer, which might be affected by primer length (longer has higher tm), primer GC content and overall base pair composition (more GC, higher temp)

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 requires primer design but can cut pretty much wherever you design

Restriction digest uses enzymes who have specific restriction sites

PCR is preferable for more novel cases, and restriction enzyme digest is for more well known plasmids with clear cut sites.

1. Why does the PvuII digest require CutSmart buffer?

The company created enzymes that operate efficiently under a standardized set of conditions, which are all present in the CutSmart buffer

1. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?