Week 6 Homework
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Week 6 Homework

  1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
    • Phusion DNA Polymerase: high-fidelity polymerase with 3—>5’ exonuclease activity for accurate DAN replication
    • dNTPs: provides nucleotides (A, T, C, G) for DNA synthesis
    • MgCl2: cofactor () for polymerase activity, stabilizing the DNA structure
    • Buffer: maintains optimal pH and ionic conditions for the reaction
    • Enhancers/stabilizers: improve enzyme performance and PCR efficiency
  2. What are some factors that determine primer annealing temperature during PCR?
    • GC content: higher GC content requires higher annealing temperatures
    • Primer length: longer primers generally require higher annealing temperatures
    • Salt concentration: affects primer binging stability
    • Primer-template complementary: mismatches lower the optimal annealing temperatures
    • Melting temperature calculation: annealing temperature is ~5C below primer melting temperature
  3. 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.
Feature
PCR
Restriction Enzyme Digest
Function
amplifies DNA
cuts DNA at specific sites
Enzyme used
DNA polymerase (Phusion)
restriction endonuclease
Sequence specificity
defined by primers
defined by restriction site
Fragment ends
can be designed (blunt/sticky)
sticky or blunt, enzyme-dependent
Best used when
amplifying specific DNA
cloning or fragmenting DNA
  1. Why does the PvuII digest require CutSmart buffer?

    2. The CutSmart buffer provides the necessary pH, salt, and Mg²⁺ conditions for PvuII activity. It optimizes enzyme specificity and efficiency, reducing star activity (non-specific cutting).

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

    3. Primer design: include 20-40 bp homologous overlaps

    High-fidelity PCR: ensure error-free amplification

    Proper restriction enzyme digest: if digestion is used, ensure correct fragment ends

    Gel purification: removes unwanted fragments

    Sequence verification: confirm that the sequence matches the cloning vector design

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

    4. Heat shock method: briefly heating the cells at 42C creates temporary membrane pores

    Electroporation: high-voltage pulses disrupt membranes, allowing DNA to enter

    Calcium chloride treatment: makes the membrane more permeable to plasmid uptake

  4. 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!

      5. Golden Gate Assembly uses TypeIIS restriction enzymes (Bsal) that cut outside their recognition sites, allowing seamless ligation of DNA fragments in a single reaction.

      Steps:

    1. design DNA fragments with matching overhangs
    2. digest with type IIS enzyme to create unique sticky ends
    3. ligate DNA fragments in a single reaction
    4. transform into bacteria for cloning

      5. Advantages:

    5. efficient and precise: scar-free assemblies
    6. one-pot reaction: no need for separate digestion and ligation steps
    7. modular: allows multi-fragment assembly