1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
   1. Phusion DNA polymerase is the enzyme that replicates the DNA
   2. Phusion Buffer keeps the pH at appropriate levels and provides optimal environmental conditions to keep everything stable
   3. dNTP are single DNA nucleotides that will be used during DNA replication as building blocks
2. What are some factors that determine primer annealing temperature during PCR?
   1. The two main factors are CG content and primer length. Other factors include various ion concentrations such as Potassium and Sodium concentrations.
   2. DMSO (Dimethyl Sulfoxide) has multiple roles, including the prevention of secondary structure formation, and lowers annealing temperature.
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. In PCR, DNA is heated up to denatured it. Specialized primers attach to the denatured DNA. The temperature is then lowered to allow a polymerase to replicate the DNA. The replicated DNA is a linear fragment.In Restriction enzyme digest, a specialized enzyme is used to snip the DNA at a particular recognition site (remember about palindromic DNA.) Restriction enzyme digest is much simpler, requiring only one step. Because of the repeated nature of PCR, it requires the use of a thermocycler. PCR is useful when you need to amplify a particular sequence, especially if you only have a small concentration. In addition, PCR can be used in combination with gel electrophoresis to detect a particular gene or DNA sequence. Yet another application of PCR is to randomly introduce mutations into a DNA sequence. Restriction Enzyme Digest is used during cloning, to linearize a plasmid in preparation for transformation. It is also used to make recombinant DNA, allowing you to cut and paste different DNA fragments together.
4. Why does the PvuII digest require CutSmart buffer?
   1. It helps keep the pH at 7.9, which is where Pvull is most efficient. It also include Mg ions, which Pvull uses as a cofactor.
5. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
   1. Make sure that the ends of the sequence have the correct overlap length and nucleotides.
6. How does the plasmid DNA enter the E. coli cells during transformation?
   1. Plasmid DNA enters the E. COLI through holes in the cell wall. Calcium ions on the competent cell attract the DNA to the cell wall, increasing transformation efficiency.
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. Biobrick assembly is a modular assembly method, used widely in iGem and for prototyping genetic circuits, in which each plasmid is identical except for small sequences in the middle which will be connected together. On the upstream side of the sequence, there are two restriction sites, Eco-RI and XbaI, while on the downstream side there are SpeI and PstI. There are multiple ways to cut the fragments up tog et different desired outputs (such as inserting fragment A into plasmid B, fragment B into plasmid A, or fragment A and fragme t B into a third plasmid C.) No matter the case, the steps are similar. Restriction enzymes are used to target the specific restriction sites on each plasmid, the results are purified, and then finally, they are mixed and ligated. Note that the sticky end from S ligates to the sticky end of X. This is possible because the restriction sites were intentionally chosen to have complementary sticky ends.