Week 6: Genetic Circuits

DNA Assembly

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

    This Master Mix is used to increase the speed and yield of the PCR product, with accurate DNA sequences maintained for DNA replication for testing. The HF buffer variant contains DNA polymerase, deoxynucleotides, buffer, and magnesium dichloride.

    source: https://www.neb.com/en-us/products/m0531-phusion-high-fidelity-pcr-master-mix-with-hf-buffer?srsltid=AfmBOoq3xHVgYXqvCn6vETXyCMTKGo4MNvB1Zs6M25yaSdNrW9j4dHWK

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

    Annealing temperatures are determined by the melting temperature of the selected primers. Some factors that determine the melting point include the number of nucleotides present in the DNA oligo (the short single strand piece of DNA for this experiment).

    source: https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-cycling-considerations.html

  3. 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.

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

    You can check that the product has the right DNA concentration for the Gibson assembly using the nanodrop after purification.

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

    The E.coli cells are thermally shocked. First they are kept cold on ice, then heat shocked for a few minutes, before

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

    1. Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).

      Golden Gate Assembly works by

    2. Model this assembly method with Benchling or Asimov Kernel!