Homework - Week 6

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, both in terms of protocol as well as when one may be preferable to use over the other.
4. Why does the PvuII digest require CutSmart buffer?
5. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
6. How does the plasmid DNA enter the E. coli cells during transformation?
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!

Resources

Primer Design: Supplemental to Gibson Assembly Recitation

Introduction to Gibson Assembly: https://www.youtube.com/watch?v=tlVbf5fXhp4

More from NEB here: https://www.neb.com/ja-jp/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/gibson-assembly