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

Phusion High-Fidelity PCR Master Mix is a 2X ready-to-use mix that contains several key components, each with a specific purpose:

• Phusion DNA Polymerase: This is the enzyme responsible for synthesizing new DNA strands. Its high-fidelity and proofreading activity (3′→5′ exonuclease activity) help minimize errors during amplification.
• dNTPs (deoxynucleoside triphosphates): These are the building blocks for new DNA synthesis. They provide the necessary nucleotides (A, T, G, C) for the polymerase to extend the DNA strand.
• Reaction Buffer (including MgCl₂): The buffer maintains an optimal pH and ionic environment for the enzyme. Magnesium ions (Mg²⁺) serve as essential cofactors that enhance enzyme activity and stability.
• Additional Stabilizers and Proprietary Additives: Many master mixes include compounds such as BSA (bovine serum albumin) and other proprietary components. These help stabilize the enzyme, reduce potential inhibitors, and improve overall reaction efficiency.

Phusion DNA Polymerase is engineered for high-fidelity DNA amplification, significantly reducing errors during PCR. This enhanced accuracy is primarily due to its intrinsic 3′ to 5′ exonuclease activity, which serves as a proofreading mechanism. Here's how this activity contributes to minimizing errors:

Proofreading Mechanism:

• 3′ to 5′ Exonuclease Activity: As the polymerase synthesizes new DNA strands, it continuously monitors the incorporation of nucleotides. If an incorrect nucleotide is added, the enzyme detects this mismatch and removes it by cleaving the erroneous nucleotide from the 3′ end. This allows the polymerase to replace it with the correct nucleotide, ensuring the accuracy of the newly synthesized DNA strand. PMC

Comparison with Other Polymerases:

• Error Rates: Phusion DNA Polymerase exhibits an exceptionally low error rate, approximately 50 times lower than that of Taq DNA polymerase and six times lower than Pfu DNA polymerase. This high fidelity makes it particularly suitable for applications where accuracy is critical, such as cloning, sequencing, and mutagenesis. Wikipedia+3Fisher Scientific+3Thermo Fisher Scientific - US+3

Structural Enhancements:

• Fusion with DNA-Binding Domain: Phusion DNA Polymerase is a fusion of a Pyrococcus-like proofreading enzyme with a DNA-binding domain. This unique structure enhances the enzyme's processivity—the number of nucleotides added per binding event—resulting in faster extension rates and higher yields, all while maintaining high fidelity.

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

   The annealing temperature (Tₐ) in PCR is critical for the specificity and efficiency of primer binding to the DNA template. Several factors influence the optimal Tₐ:

   

   1. Primer Melting Temperature (Tₘ):The melting temperature is the point at which half of the DNA duplex dissociates into single strands. Primers with higher Tₘ values require higher annealing temperatures to bind specifically to the template. A common guideline is to set the Tₐ approximately 5°C below the Tₘ of the primers.

   2. Primer Length and Base Composition:Longer primers and those with higher GC content have increased Tₘ due to more hydrogen bonds, necessitating higher Tₐ. Conversely, primers rich in AT pairs have lower Tₘ and require lower Tₐ.

   3. Primer-Template Mismatches: Mismatches between the primer and template can destabilize binding. Higher Tₐ can reduce nonspecific binding, while lower Tₐ may tolerate mismatches, leading to nonspecific amplification.

   4. Salt Concentration in the Reaction Buffer:Ions like Mg²⁺ stabilize primer-template duplexes. Higher salt concentrations can increase Tₘ, allowing for higher Tₐ, whereas lower concentrations might necessitate reduced Tₐ.

   5. Primer Concentration:Elevated primer concentrations can promote nonspecific binding, especially at lower Tₐ. Adjusting primer concentration alongside Tₐ is essential for optimal specificity.

   6. PCR Product Length:Longer amplicons may require adjustments in Tₐ to ensure efficient and specific amplification.

   Balancing these factors is crucial. An annealing temperature too low can result in nonspecific binding, while one too high may reduce primer binding efficiency, leading to decreased yield. Empirical optimization or gradient PCR is often employed to determine the optimal Tₐ for specific primer-template systems.

2. 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 Digest
   Specificity	High (primer dependent)	High (enzyme site dependent)
   Customization	Highly customizable, allows mutations	Limited customization (fixed sites)
   Procedure Complexity	Moderate (primer design critical)	Low (simple mixing and incubation)
   Flexibility	Very high (varied fragment sizes/overlaps)	Lower (dependent on restriction sites)
   Efficiency for mutations	Excellent	Poor (does not introduce mutations)
   Typical Application in Protocol	Creating amplicons for Gibson Assembly	Generating linear backbone from plasmid

3. Why does the PvuII digest require CutSmart buffer?

   The PvuII digest requires CutSmart buffer because this buffer provides the optimal conditions necessary for the activity and specificity of the restriction enzyme PvuII. Specifically:

   • Optimal Ionic Strength and pH:

     CutSmart buffer maintains the ideal pH (~7.9 at 25°C) and ionic conditions necessary for PvuII enzymatic activity. Restriction enzymes like PvuII are sensitive to changes in pH or salt concentration, and incorrect conditions can significantly reduce or abolish enzymatic activity.

   • Divalent Cations (Mg²⁺):

     CutSmart buffer includes magnesium ions (Mg²⁺), an essential cofactor required for the catalytic activity of most restriction enzymes, including PvuII. Without Mg²⁺, the enzyme cannot cleave DNA.

   • Stability and Fidelity:

     Using CutSmart buffer enhances enzyme stability and fidelity, ensuring precise and clean DNA cleavage, thus improving efficiency in downstream applications like Gibson Assembly or ligation.

   • Universal Buffer Compatibility:

     CutSmart buffer (from New England Biolabs) is a universal buffer optimized for a wide variety of restriction enzymes, simplifying workflows when multiple enzyme digestions might be required.

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

   1. Verify Fragment Ends and Overlaps:

   • Correct Overlaps:

     Ensure each fragment has overlapping sequences of 20–40 nucleotides at each junction. Gibson Assembly relies on these complementary overlaps created either by PCR primers or strategic digestion.

   • Overlap Orientation:

     Confirm fragments are oriented correctly (5'→3') so complementary overlaps align appropriately.

   2. PCR Primers Design Validation:

   • Primer Length and Overhangs:

     Check that primers contain 18–22 bp core binding regions, plus 20–22 bp overhangs complementary to the adjacent fragment (e.g., plasmid backbone).

   • Tm Compatibility:

     Verify melting temperatures (Tm) of primers are within 5°C of each other (optimal range usually 52–58°C) to ensure specificity during PCR.

   • Secondary Structures:

     Confirm primers do not form stable secondary structures (hairpins or dimers), as these can interfere with PCR efficiency or Gibson Assembly annealing steps.

   3. Restriction Digest Validation:

   • Correct Enzyme & Buffer Usage:

     Confirm correct restriction enzymes (such as PvuII) and optimal buffer conditions (CutSmart Buffer) were used.

   • Fragment Purity:

     Run a diagnostic agarose gel to verify that restriction digestion produces a single, clean, linear band at the expected size.

   4. Diagnostic Gel Electrophoresis:

   • Run fragments (both PCR products and digested DNA) on agarose gel electrophoresis:
     • Ensure PCR fragments are the correct predicted sizes.
     • Confirm restriction digest fragments yield clean bands with no visible contamination or unexpected bands.

   5. DNA Purification & Quantification:

   • Purify DNA fragments using kits (e.g., Zymo DNA Clean & Concentrator) to remove contaminants (enzymes, primers, nucleotides, salts).
   • Measure concentrations with Nanodrop/Qubit to ensure adequate yields (ideally >30 ng/µL).

   6. Gibson Assembly Ratio Calculation:

   • Use the recommended molar ratio (~2:1 insert:vector) for optimal assembly efficiency.
   • Calculate precise amounts of each fragment to ensure efficient and correct Gibson assembly.

   7. Control Gibson Reaction (Recommended):

   • Include a negative control (no insert or no backbone) and a positive control (known working plasmid/fragments) to verify Gibson Assembly conditions are correct and effective.

   8. Sequencing Validation Post-Assembly:

   • After Gibson Assembly and bacterial transformation, verify colonies via sequencing (Sanger sequencing) or colony PCR to confirm correct assembly and desired mutations/inserts.

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

   Plasmid DNA enters E. coli cells during transformation through temporary pores created in the bacterial membrane. Two main methods are:

   • Heat Shock: Calcium chloride treatment followed by brief heating at 42°C temporarily destabilizes cell membranes, allowing plasmids to diffuse inside.
   • Electroporation: A short, high-voltage electric pulse opens transient membrane pores, through which plasmids enter cells rapidly.

   After both methods, cells recover in nutrient-rich media (SOC), allowing membrane repair and expression of plasmid-encoded antibiotic resistance. Transformed cells then survive antibiotic selection and form colonies.

3. 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!

Resources

Primer Design: Supplemental to Gibson Assembly Recitation