Genetic Circuits Part 1

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

Components: Phusion DNA Polymerase (low error rate), nucleotides (A, T, C, G without U), reaction buffer (ensure maximum enzyme activity);

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

Each primer has a different annealing temperature determined by the environment and by the nucleotides that make up the primer

  1. 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 makes strands by attaching primers with tails that hang freely. The result is a DNA sequence made of the original one + the tails of the primers that were added. Restriction enymes cut DNA and the resulted sequence part of the original sequence.

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

The sequences need to have identical ends in order to be cut and ligated.

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

The E.coli is thermally or electrically stimulated to accept the vector by creating holes in the membrane. This often kills the bacteria..There are newer methods that use really small tubes to create holes that do not damage the bacteria as much as the two previous methods.

  1. 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!

Golden Gate assembly is a highly efficient DNA cloning method. It uses type II enzymes that “cut” DNA consequent to recognized nucleotide sequence (usually palindromic), mostly after 4-5 nucleotides-post recognition site; and are dependent of Mg2+ as a cofactor. They act on both the plasmid and fragment pretty much the same; thus, the digested insertion fragment covalently binds to plasmid overhang complementary DNA strand.

Compared to Gibson a. it doesn’t require polymerases to build up lacking DNA strand fragments, but it does usually include ligase enzymes to ensure vector stability and successful annealing. G.G. assembly is preferred when working with many insertion fragments (30-50) because of its simultaneous activity of ligases and restriction enzymes.