1. 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: It offers both high fidelity and robust performance and it is an ideal choice for cloning and can be used for long or difficult amplicons. Its function is to synthesize new DNA strands from a template, using nucleotides.

-Deoxynucleotide triphosphates (dNTPs): They are the essential building blocks of DNA synthesis during amplification.

-Reaction buffer: It serves as a medium for the reaction and provides the necessary pH and salt concentration to ensure efficient DNA amplification. In addition, it contains MgCl2, which is required for the activity of Phusion DNA polymerase.

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

The primer annealing temperature is dependent on the base composition (the proportion of A, T, G and C nucleotides), primer concentration and ionic reaction environment.

1. 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.

The PCR technique can be used when restriction enzymes are not available, and the specific region to amplify is well-known. However, it is important to consider the use of a high-fidelity polymerase and ensure that the sequence to be amplified is not too long.

On the other hand, digestion with restriction enzymes is commonly used for cloning due to the generation of sticky or blunt ends. However, to use this method, specific restriction enzymes are required, and it may also require an additional purification step compared to PCR.

1. Why does the PvuII digest require CutSmart buffer?

The PvuII enzyme is considered a high-fidelity enzyme that requires only a single buffer, such as CutSmart Buffer, to function optimally and achieve 100% activity.

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

We can ensure that the PCR products and the linear blunt-ended backbone fragment generated from the digestion of the pUC19 plasmid with the PvuII restriction enzyme will be appropriate for Gibson cloning through concentration measurement and diagnostic gel electrophoresis.

The concentration measurement using Nanodrop or Qubit should result in DNA concentrations above ~30 µg/mL.

The diagnostic gel electrophoresis is used to verify the expected size of the fragments obtained after digestion with the PvuII restriction enzyme. It is very important to first calculate the predicted digest on Benchling.

6. How does the plasmid DNA enter the E. coli cells during transformation? The most common methods for introducing plasmid DNA into bacteria are electroporation and heat shock. These methods create pores in the bacterial cell wall, allowing the plasmid DNA to enter the cells.