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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?
The Phusion High-Fidelity PCR Master Mix contains several key components that enable efficient and accurate DNA amplification:
- Phusion DNA Polymerase – A genetically engineered, high-fidelity DNA polymerase with strong 3’→5’ exonuclease (proofreading) activity. This enzyme ensures accurate DNA synthesis by correcting misincorporated nucleotides, significantly reducing the error rate compared to standard Taq polymerase.
- dNTPs (deoxynucleotide triphosphates) – These are the four DNA nucleotide building blocks (dATP, dTTP, dCTP, dGTP) that are incorporated into the growing DNA strand during polymerization.
- Optimized Reaction Buffer – This contains salts (such as KCl and (NH₄)₂SO₄), pH stabilizers, and MgCl₂, which are critical for enzyme activity and DNA strand annealing. Magnesium ions (Mg²⁺) serve as a cofactor for the DNA polymerase, ensuring proper catalytic function and stabilization of the DNA structure. The buffer is formulated to support the polymerase’s high-fidelity performance while reducing errors and improving yield.
- What are some factors that determine primer annealing temperature during PCR?
- Primer Length – Longer primers generally have a higher annealing temperature (Tm) because they form more hydrogen bonds with the template, making them more stable.
Short primers (e.g., <18 nucleotides) have lower Tm and may bind non-specifically.
- GC Content – A higher GC content increases Tm because G≡C base pairs have three hydrogen bonds, while A=T base pairs have only two.
A rough estimate is Tm increases by ~4°C for every 10% increase in GC content.
- Salt Concentration (Especially Mg²⁺) – Higher salt concentrations stabilize primer-template binding, raising the annealing temperature. Magnesium ions (Mg²⁺) help neutralize negative charges on DNA, promoting proper hybridization.
- Mismatches Between Primer and Template – Perfect matches increase Tm, while mismatches lower Tm by disrupting base pairing. This is crucial in applications like site-directed mutagenesis.
- Primer-Template Secondary Structures – Hairpins or self-dimers in primers can reduce effective binding and lower the apparent Tm. Template secondary structures (like GC-rich regions forming strong hairpins) can also affect primer binding increasing 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.
| Parameter |
PCR |
Restriction Enzyme Digest |
| Duration |
Typically 1–2 hours (depends on the number of cycles and polymerase speed) |
Usually 15–60 minutes, though some reactions may require longer incubation (e.g., star activity prevention) |
| Components Needed |
Primers (to define amplification region) + DNA polymerase + dNTPs + Buffer + Template DNA |
Restriction enzyme(s) + Buffer + Template DNA + (sometimes) BSA for enhanced enzyme activity |
| Equipment Needed |
Thermocycler (for repeated heating and cooling cycles) |
37°C incubator or water bath (most enzymes work at 37°C, though some require different conditions) |
| Output DNA |
Blunt or sticky ends, depending on primer design (sticky ends require engineered primers) |
Blunt or sticky ends, depending on the restriction enzyme used |
|
| Preferred Use | When amplifying a specific sequence from a template (e.g., gene cloning, site-directed mutagenesis, diagnostic testing) | When cutting DNA at specific recognition sites (e.g., subcloning into vectors, creating recombinant DNA, analyzing mutations) |
| Flexibility | Can amplify any sequence with designed primers | Limited to cutting at specific recognition sites present in the DNA |
| Sequence Specificity | Defined by primer binding sites | Defined by restriction site recognition (typically 4–8 bp palindromic sequences) |
| Sequence Modification | Can introduce mutations or modifications through primer design | Does not modify sequence unless additional steps (e.g., ligation) are performed |
- Why does the PvuII digest require CutSmart buffer?
PvuII requires CutSmart buffer because it provides optimal conditions, including the right pH, ionic strength, and Mg²⁺ concentration, for efficient DNA cleavage at its recognition site. This buffer ensures enzyme activity and minimizes the risk of non-specific cuts, leading to accurate and complete digestion.”
- How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?