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

The Phusion High-Fidelity PCR Master Mix includes a high-fidelity DNA polymerase for accurate DNA synthesis, an optimized reaction buffer to support enzyme activity, and dNTPs as the necessary building blocks for new DNA strands. These components allow you to perform PCR by simply adding your specific DNA template, primers, and sterile water.

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

Primer annealing temperature is influenced by the melting temperature (Tm) of the primers, their GC content, primer length, buffer composition, salt concentration, and the presence of additives like DMSO. These factors affect how efficiently and specifically the primers bind to the DNA template.

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 uses specific primers and a polymerase to selectively amplify a DNA region, producing many copies of a target sequence and allowing for easy modification or amplification even without restriction sites. Restriction enzyme digest, on the other hand, uses enzymes to cut DNA at defined sequences, which is simple and effective if the template already contains the necessary sites. PCR is preferable for amplifying or modifying DNA, while restriction digest is ideal for cutting existing plasmids at known sites for cloning.

Why does the PvuII digest require CutSmart buffer?

CutSmart buffer is specifically formulated to provide the optimal ionic strength and pH for the activity of many restriction enzymes, including PvuII. Using this buffer ensures efficient and reliable digestion.

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

You can design overlapping ends for each fragment, verify the sizes and purity of your DNA by gel electrophoresis, and use in silico simulations to check that the fragments will assemble correctly during Gibson cloning.

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

During heat shock transformation, the rapid temperature change creates temporary pores in the E. coli cell membrane, allowing plasmid DNA to enter the cell. The precise molecular details are not fully understood, but the process is highly effective for introducing DNA into bacteria.

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!

Golden Gate Assembly is a cloning method that uses Type IIS restriction enzymes, like BsaI, which cut outside their recognition sites to create custom overhangs on DNA fragments. These overhangs guide the fragments to assemble in a specific order and orientation. T4 DNA ligase is added to join the fragments together in a single-tube reaction, while the enzyme repeatedly digests any incorrect assemblies. The process cycles between digestion and ligation steps, ensuring only the correctly assembled DNA remains. This method is efficient for assembling multiple fragments at once without leaving extra sequences (scars) between them. Designing the fragments with matching overhangs is key to successful assembly. The process can be visualized and planned using software tools like Benchling.