Week 6 HW: Genetic Circuits Part I: Assembly Technologies

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

The components in the Phusion High-Fidelity PCR Master Mix are Phusion DNA Polymerase, deoxynucleodites, reaction buffer.

-Phusion DNA Polymerase-copies the DNA

-Reaction buffer-maintains optimal biochemical proprieties

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

-specific annealing enzyme use and their optimal catalysing activity temperature;

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.

PCR amplifies DNA, while r.e.d. cut DNA. Mainly, PCR is used to get more genetic material to work with- either test or express. R.e.d. is used in DNA testing and gene insertion in Gibson assembley and others.

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?

Electroporation or thermic shock.

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

Gibson Assembly is a cloning method that sticks together multiple PCR fragments to make a single circular plasmid, without needing traditional restriction enzymes or separate ligation steps.

Each fragment is designed so that its ends include 20–40 base‑pairs of matching sequence with the neighboring fragment, forming overlapping “sticky ends” in the right order.

A DNA polymerase fills in any gaps left after annealing, and a DNA ligase seals the nicks, giving a fully closed, double‑stranded DNA molecule. Because the reaction is isothermal and the overhangs are designed computationally, Gibson can assemble many fragments in one step and is very flexible in what kind of vector you can use.