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

Ans:

Phusion DNA Polymerase: A high-fidelity enzyme for accurate amplification, with error rates of $4.4×10^{−7}$ (HF Buffer) or $9.5×10^{−7}$(GC Buffer)
HF or GC Buffer:
- HF Buffer: Default for high-fidelity amplification, optimized for standard templates
- GC Buffer: Enhances performance on GC-rich or complex templates
dNTPs: Pre-mixed deoxynucleotides (200 µM final concentration) for DNA synthesis
MgCl₂: Pre-optimized concentration (1.5 mM final) to stabilize DNA-polymerase interactions

The mix reduces setup steps, ensuring consistent reaction conditions for efficient amplification.

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

Ans: Key factors include:

Primer Melting Temperature (Tm): Calculated using formulas like $Tm=4(G+C)+2(A+T)Tm=4(G+C)+2(A+T)$ or $Tm=81.5+16.6(log⁡[Na+])+0.41(\%GC)−675/primer length$
GC Content: Higher GC content increases Tm due to stronger hydrogen bonding
Buffer Composition: Additives like DMSO or betaine lower Tm, requiring adjusted annealing temperatures.
Template Complexity: GC-rich or secondary structures may necessitate higher annealing temperature

Optimal annealing temperature is typically 3–5°C below the primer Tm

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.

Ans: PCR vs. Restriction Digest: Comparison

Aspect	PCR	Restriction Digest
Protocol	Amplifies DNA using primers and thermal cycling. Requires DNA polymerase, dNTPs, and buffer	Cuts DNA at specific sites using restriction enzymes. Requires enzyme, buffer, and optional BSA
Output	Generates copies of a specific DNA region.	Produces linear fragments with sticky/blunt ends.
Use Case	Preferred for unknown sequences or low DNA quantities.	Ideal for cloning known sequences with restriction sites
Time	1–3 hours (thermal cycling).	15–60 minutes (single-step digestion)

Why does the PvuII digest require CutSmart buffer?

Ans: CutSmart Buffer ensures:

Optimal Enzyme Activity: Maintains pH and ionic conditions for PvuII-HF efficiency
Reduced Star Activity: Minimizes non-specific cleavage
Compatibility: Allows simultaneous use of multiple enzymes without buffer changes
BSA Inclusion: Stabilizes enzymes and prevents aggregation

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

Ans: Ensuring DNA Suitability for Gibson Cloning

Overlap Design: Fragments must have 20–40 bp homologous overlaps for assembly
High-Fidelity PCR: Use Phusion polymerase to minimize mutations
Purification: Remove enzymes, salts, or primers that inhibit ligation
Digestion Validation: Confirm clean restriction enzyme cuts via gel electrophoresis

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

Ans: E. coli cells are made chemically competent via calcium chloride treatment, neutralizing their membrane charge. A heat shock (42°C for 45 seconds) creates pores in the membrane, allowing plasmid DNA uptake. After shock, cells recover in nutrient-rich media before plating

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!

Ans: Golden Gate Assembly

Method: Uses Type IIS restriction enzymes (e.g., BsaI) to cut outside recognition sites, generating unique overhangs. Fragments with complementary overhangs ligate seamlessly in a single reaction

Steps:

Design fragments with 4–6 bp overhangs.
Digest and ligate in one tube using enzyme + ligase.
Cyclic temperature shifts (37°C for digestion → 16°C for ligation) enhance efficiency

Benchling Modeling:

Use the combinatorial tool to design fragments with compatible overhangs.
Simulate digestion/ligation steps and validate construct design

Advantages:

Scarless, multi-fragment assembly (up to 35 fragments)
No residual restriction sites in the final product

By Rob Hurt - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=88975448

Resources

Primer Design: Supplemental to Gibson Assembly Recitation

Introduction to Gibson Assembly: https://www.youtube.com/watch?v=tlVbf5fXhp4