Lab Followup

1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
   1. Some key components are the high fidelity DNA polymerase from Thermus aquaticus, the buffer solution with optimal conditions (pH,ionic force, cofactors) for enzyme work,dNTPs.
2. What are some factors that determine primer annealing temperature during PCR?
   1. Some factors could be: the primer length (longer primers requires higher temperatures to be separated from their template), the same occurs with CG content (the higher the more stable) and salt concentration (higher salt concentration requires higher temperatures).
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.
   • PCR generates DNA fragments through enzymatic replication, while restriction enzyme digest cuts DNA at specific recognition site
   • PCR can amplify specific DNA sequences, while restriction enzyme digest can generate fragments with defined ends but without increasing the DNA molecules.
   • PCR is more versatile and can amplify DNA from diverse sources, while restriction enzyme digest is limited to DNA sequences containing specific recognition sites.
   • PCR can introduce mutations or errors during amplification, while restriction enzyme digest typically does not alter the DNA sequence.
   • So PCR is the best option when big DNA quantities are needed for downstream applicationes. While restriction enzyme digest generates defined ends useful for subsequent cloning or manipulation.
4. Why does the PvuII digest require CutSmart buffer?
   1. To ensure optimal enzyme activity.
5. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
   1. The first thing to take into account is a correct experimental design, GIbson assembly requires overlapping ends that can be generated through PCR; the generated DNA fragments also need to have sequences that allow them to be integrated into plasmids or other vectors. Once the fragments are generated, their quality can be assessed using Southern blots, and DNA quantification methods.
6. How does the plasmid DNA enter the E. coli cells during transformation?
   1. Competent cells with altered cell wall permeability are needed for transformation. This competent cells are incubated with the DNA we want to introduce and then transient pores in the membrane are induced by thermic/chemical/electro shock.

Golden Gate Assembly is a molecular biology technique used for the assembly of multiple DNA fragments into a larger DNA construct, typically a plasmid. It is based on the use of type IIS restriction enzymes, which cut DNA sequences outside their recognition site, leaving defined overhangs that can be precisely engineered to facilitate assembly.