Week 6 HW: Genetic Circuits Part 1 : Assembly Technologies

DNA ASSEMBLY

  1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
  • High-Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification.
  • Phusion High-Fidelity DNA Polymerase offers both high fidelity and robust performance, and thus can be used for all PCR applications.
  • Its unique structure, a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain, increases fidelity and speed. Phusion DNA Polymerase is an ideal choice for cloning and can be used for long or difficult amplicons.
  • With an error rate >50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase,
  • Phusion is one of the most accurate thermostable polymerase available. Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products.
  • Phusion High-Fidelity PCR Master Mix with HF Buffer is a 2X master mix consisting of Phusion DNA Polymerase, deoxynucleotides and reaction buffer that has been optimized and includes MgCl2. All that is required is the addition of template, primers and water.

Phusion High-Fidelity PCR Master Mix is a 2X concentrated, ready-to-use solution containing Phusion DNA Polymerase, optimized reaction buffer (HF or GC), MgCl2, and dNTPs. It provides high-fidelity, high-speed, and robust amplification, designed for blunt-end cloning. Only template DNA, primers, and water are needed for reaction setup.

Function : Phusion DNA Polymerase : Optimized reaction buffer (HF or GC) : MgCl2 : dNTPs :

  1. What are some factors that determine primer annealing temperature during PCR? Primary factors that determine the optimal primer annealing temperature :
  • Primer Melting Temperature
  • Primer Length
  • GC Content
  • Salt Concentration Mg2+ and Monovalent Cations
  • Primer Concentration
  • Primer-Dimer and Hairpin Formation
  • Mismatches with the Template
  • Note : If you’re doing electrophoresis and the result is smear or no band, is likely too high annealing temperature (try lowering it). If the results is multiple bands (Non-specific), is likely too low annealing temperature (try raising it).
  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.
  2. How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
  3. How does the plasmid DNA enter the E. coli cells during transformation?
  4. 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!