Week 6 HW: Genetic Circuits Part I: Assembly Technologies

Assignment: DNA Assembly

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

as per ¹

• High fidelity DNA polymerase (50 times more accurate than “standard” Thermus aquaticus DNApoly)
• Phusion HF buffer
• Mg²⁺ as 1.5mM MgCl₂ for DNApoly to function
• dNTPs

What are some factors that determine primer annealing temperature during PCR?

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.

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

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

Describe another assembly method in detail (such as Golden Gate Assembly)

Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).

Model this assembly method with Benchling or Asimov Kernel!