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

Polymerase: grabs nucleotides floating by and sticks them onto the naked single strand. The polymerase for PCR can survive being heated to near boiling temperatures.

Nucleotides: lots of nucleotides to build new complementary strands.

Other salts and chemicals: needed for the polymerase to work.

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

The length of the primer: longer primers melt at a higher temperature (just like fats or hydrocarbons).

I think some of the nucleotide bases (G & C?) bind more strongly, so if you have more of those, the annealing temperature also goes up.

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.

PCR can amplify: starting with just a few strands of long DNA, you can make billions of copies of a small region of it. It’s useful for things like forensics and medical diagnostics, when you’re starting with only a small amount of the DNA you want.

Restriction enzymes chop up existing DNA, but can’t make more copies of it. You’d want this when chopping up a plasmid, because the restriction enzymes can break the ring. And restriction enzymes are easier to use than PCR, because you don’t need all the heating cycles, and you don’t need to design and order primers.

Why does the PvuII digest require CutSmart buffer?

The restriction enzymes need a few other chemicals to do their job. CutSmart claims to contain the chemicals needed for hundreds of restriction enzymes, so you don’t have to think about which buffer you’re using for the digests.

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

The sticky ends of the PCR-amplified inserts need to line up with the exposed ends of the digested plasmid.

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

You can shock the E. coli to make its membrane more permeable to plasmids — either with chemicals, or electricity, or heat.

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

Speculating a bit (I’m sure other students will cover Golden Gate assembly, so I want to try something more exotic) …

I wonder if you can use CRISPR for plasmid assembly. Like, use CRISPR to cut the plasmid, leaving sticky ends, then rely on homologous joining to make the insert attach to the cut plasmid.