Homework — Genetic Circuits Part I

Warning

These questions are based on the Gibson Assembly Lab and are mandatory for Committed Listeners and MIT/Harvard students.

Questions

  1. What are some components in the Phusion High‑Fidelity PCR Master Mix and what is their purpose?
  2. What are some factors that determine primer annealing temperature during PCR?
  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, including when one may be preferable.
  4. Why does the PvuII digest require CutSmart buffer?
  5. How can you ensure that the DNA sequences you digested and PCR‑ed will be appropriate for Gibson cloning?
  6. How does the plasmid DNA enter E. coli cells during transformation?
  7. Describe another assembly method in detail (e.g., Golden Gate Assembly) in 5–7 sentences with diagrams. Model this method with Benchling or a similar tool.