Week 6 HW: Genetic Circuits Part I

PART 1: Protocol questions

  1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?

The mastermix contains:

  • Phusion Hi-Fi DNA Polymerase: It is crucial for completing the amplicons generated during PCR.
  • Deoxynucleotides: The building blocks necessary for replicating DNA fragments.
  • Buffer including MgCl2: Prevents enzyme denaturation by maintaining pH at a fixed level.
  1. What are some factors that determine primer annealing temperature during PCR?

Some factors include the primer length

  1. There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other
CategoryPRCRestriction Enzymes
Protocol

  1. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?

  2. How does the plasmid DNA enter the E. coli cells during transformation?

The plasmid DNA enters the cells through a process called electroporation where, by means of an externally applied voltage, the membrane permeability increases, allowing the plasmid to enter the bacterial cytosol.

  1. Describe another assembly method in detail (such as Golden Gate Assembly) 6.1 Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online) 6.2. Model this assembly method with Benchling or Asimov Kernel!