What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
The Phusion High-Fidelity PCR Master Mix typically contains:
- Phusion DNA Polymerase: a proofreading polymerase with 3'→5' exonuclease activity, ensuring high fidelity during DNA synthesis.
- dNTPs: deoxynucleotide triphosphates serve as the building blocks for new DNA strand synthesis.
- MgCl₂: a cofactor required for polymerase activity.
- Buffer system: stabilizes pH and ionic strength, optimized for high fidelity.
- Stabilizing agents: may include BSA or other proprietary additives to enhance enzyme stability and performance.
What are some factors that determine primer annealing temperature during PCR?
Key factors include:
- Primer length: Longer primers generally have higher melting temperatures (Tm).
- GC content: Guanine and cytosine form three hydrogen bonds, increasing Tm.
- Primer sequence: Secondary structures or mismatches can affect binding and annealing temperature.
- Salt concentration: Higher salt stabilizes duplex DNA, increasing Tm.
- Concentration of Mg²⁺ and other ions: Influences the electrostatic interactions between DNA strands.
- A typical annealing temperature is 3–5 °C below the primer’s 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.
| Feature | PCR | Restriction Digest |
|---|---|---|
| Mechanism | Enzymatic amplification using primers and polymerase | Cleavage at specific recognition sites by restriction enzymes |
| Specificity | Defined by primer binding | Defined by presence of recognition sequences |
| Flexibility | Highly customizable for any sequence | Limited to sequences containing enzyme cut sites |
| Protocol Complexity | Requires thermal cycler and optimization | Simpler but requires verification of cut sites |
| Use Case | Amplify specific sequences or add overhangs/tags | Generate defined fragments for cloning or analysis |
PCR is preferable when the target needs to be modified, amplified, or lacks restriction sites. Restriction digest is useful when precise, non-mutated sequence excision is needed.
Why does the PvuII digest require CutSmart buffer?
PvuII requires CutSmart buffer because it provides the optimal ionic strength, pH (7.9), and cofactors (such as Mg²⁺) for maximal enzyme activity and stability. NEB’s CutSmart buffer is compatible with over 200 restriction enzymes, simplifying digestion protocols and reducing the need for buffer switching.
How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
To ensure compatibility with Gibson Assembly:
- Design overlapping sequences (15–40 bp) between adjacent fragments.
- Avoid internal secondary structures in overlap regions.
- Ensure high-fidelity PCR products to minimize mutations.
- Use gel purification to eliminate non-specific fragments or primer dimers.
- Verify the sequence orientation and reading frame, especially when assembling coding regions.
How does the plasmid DNA enter the E. coli cells during transformation?
During heat shock transformation, E. coli cells treated with CaCl₂ become competent by neutralizing membrane charges. A short heat pulse (usually 42°C for 30–60 seconds) creates a thermal imbalance that facilitates the uptake of plasmid DNA through transient pores in the membrane. In electroporation, a high-voltage pulse induces temporary membrane permeability, allowing DNA to enter.
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!
Golden Gate Assembly is a molecular cloning method that uses type IIS restriction enzymes (e.g., BsaI), which cut outside of their recognition sites. This allows scarless, directional assembly of multiple DNA fragments in a single reaction.
Mechanism:
- Type IIS enzymes generate custom overhangs upon digestion.
- Ligase seals these overhangs during the same reaction.
- The design ensures fragments only ligate in the correct order.
- Enzyme cycling (37°C digestion / 16°C ligation) increases efficiency.
- Golden Gate is ideal for synthetic biology, as it allows rapid and modular construction of DNA parts (promoters, genes, terminators).
