1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
   1. DNA Polymerase (to add nucleotides when building DNA), dNTPs (Deoxynucleotide Triphosphates, building blocks to add groups during DNA synthesis), Buffer Solution (maintains pH and conditions for primer binding and polymerase functions), MgCl₂.
2. What are some factors that determine primer annealing temperature during PCR?
   1. Primer sequence (length, composition, # of H bonds), primer concentration, buffer system used (salts may affect stability of DNA and affect Tm).
3. 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.
   1. PCR is for amplification of a DNA strand, while RED is for cleaving a DNA in a specific location. While the former leads to millions of copies of a DNA strand, the latter leads to several fragments based on the cleavage site.
   2. Both require a template to know which part of the DNA to copy or cut, and take ~1-2 hours. PCR requires thermal cycling, while RED requires an incubator or constant temperature.
   3. PCR is useful when you start with low amounts of DNA to amplify, while RED is useful when you have larger amounts, and known restriction sites, or want a simpler protocol without thermal cycling.
4. Why does the PvuII digest require CutSmart buffer?
   1. The restriction enzyme Pvull requires this buffer as it provides the optimal pH, salt concentration, and cofactors (like Mg ions) that allows for efficient cleavage.
5. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
   1. Ensure the primers have significant (20-40 bp) overlap.
   2. Run gel electrophoresis to confirm PCR and digest products for size.
   3. Purify components to remove any enzymes, primers, buffer component, etc. before Gibson Assembly.
6. How does the plasmid DNA enter the E. coli cells during transformation?
   1. E. Coli cells are treated to affect their membrane, and allow DNA to stick and eventually pass through more easily. The DNA is mixed with the cells on ice, and then there is a heat shock step, which creates pores or openings allowing the DNA to enter the cells. They are then cooled back down and recover for some time.
7. Describe another assembly method in detail (such as Golden Gate Assembly) 5 - 7 sentences w/ diagrams (either handmade or online). Model this assembly method with Benchling or a similar tool!
   1. In Golden Gate Assembly, another cloning method, restriction enzymes and DNA ligase are used to assemble multiple DNA fragments in a defined order and orientation, in one single reaction. Unlike typical restriction enzymes, the IIS enzymes used in GGA cut outside their recognition sites, generating custom overhangs that can be designed to be unique and complementary between adjacent fragments. During the reaction, the enzyme cuts and releases the recognition sites, allowing the DNA fragments to be ligated seamlessly without leaving extra bases. The reaction cycles between digestion and ligation steps, ensuring high efficiency and specificity of assembly. This method is especially useful for assembling multiple fragments simultaneously and is widely used in synthetic biology and modular cloning projects. [I used ChatGPT to help me answer this.]

https://www.snapgene.com/guides/golden-gate-assembly