Homework

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> These homework questions are based on the Gibson Assembly Lab! Mandatory for both CL and MIT/Harvard students.

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<aside> <img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" /> Key Links: https://docs.google.com/document/d/1_aSV7w8iRYc3EDmbueJ_hSEGy_jHLDfxT2wAezEtC4c/edit?tab=t.0#heading=h.a157u2dx9dhb

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

Phusion High-Fidelity PCR Master Mix (2X Phusion PCR Master Mix) contains 0.04 U/μL Phusion HighFidelity DNA Polymerase, 2X Phusion HF Buffer (in F-531) or 2X Phusion GC Buffer (in F-532), and 400 μM of each dNTP.

  1. A high-fidelity DNA polymerase with 3'→5' exonuclease (proofreading) activity, ensuring low error rates during DNA synthesis.
  2. HF buffer contains optimized salt composition to provide an ionic environment that maximizes Phusion polymerase fidelity and processivity, magnesium ions which are essential cofactor for polymerase activity and also ensures proper DNA synthesis, pH stabilizers to maintain an optimal pH range (around pH 8.5-9.0) to enhance enzyme function and DNA stability.
  3. GC buffer contains higher salt concentration which enhances polymerase activity on difficult templates by disrupting strong GC base pairing and DMSO (Dimethyl Sulfoxide) which helps to destabilize GC-rich regions and secondary structures, reducing template rigidity.
  4. A balanced mix of dATP, dTTP, dGTP, and dCTP, or dNTPs, serving as the building blocks for new DNA strand synthesis.

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

The primer annealing temperature during PCR is a critical parameter that affects specificity and efficiency. Several factors determine the optimal annealing temperature:

  1. Annealing temperature (Ta) is at least 2 to 3 degrees below the melting temperature (Tm).
  2. Longer primers (≥20 bp) have higher Tm, requiring higher Ta.
  3. GC content increases Tm due to stronger GC triple hydrogen bonding. Therefore Ta increases.
  4. Higher Mg²⁺ and salt stabilize primer-template binding, allowing lower Ta. Too much magnesium ion can lead to non-specific binding.
  5. GC-rich templates require higher Ta to prevent secondary structures. AT-rich regions require lower Ta due to weaker AT double bonds.