Homework#6
- What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
Phusion is a master mix useful for high-fidelity DNA amplification, its components are described below:
- Phusion DNA polymerase: a polymerase with exonuclease function in the 3'->5' direction and 5'->3' polymerase, making it 50X more efficient than alternatives such as Taq.
- dNTPs: nucleotides that function as building blocks to form new DNA strands.
- Buffer: maintain proper pH in the reaction and have additional components that can help stabilize the reaction, such as MgCl2.
Necessary but not supplemented:
- Nuclease-free water: helps adjust the reaction volume to the proper one.
- Primers (reverse and direct): short DNA sequences that direct the polymerase to the site to be amplified.
DNA: sample to be amplified and used as a template
Sources:
https://www.neb.com/en/products/m0531-phusion-high-fidelity-pcr-master-mix-with-hf-buffer?utm_
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012771_Phusion_HiFi_PCR_MasterMix_100rxn_UG.pdf?utm_
- What are some factors that determine primer annealing temperature during PCR?
Factors:
- primer length: longer primers have a higher alignment temperature due to higher binding stability (H bonds) and it is generally suggested that primers be long enough to improve specificity.
- G+C content: higher G and C content increases the alignment temperature because these bases form three hydrogen bonds, forming more stable and stronger structures that require more energy to separate
- salt concentration: a higher concentration of these ions allows higher temperature alignment by stabilizing primer-DNA binding.
- Polymerase conditions: these require different optimal temperatures for alignment.
Lorenz T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. Journal of visualized experiments : JoVE, (63), e3998. https://doi.org/10.3791/3998
- 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.
- Why does the PvuII digest require CutSmart buffer?
The aforementioned buffer allows for the efficient and correct functioning of PvuII specifically. This optimizes sample processing, as the buffer has the highest level of enzyme compatibility with 100% activity in its presence.
New England Biolabs. (2017). Restriction Endonucleases: A Guide to Enzyme-Based DNA Manipulation. Retrieved from https://www.neb-online.de/wp-content/uploads/2017/08/New-England-Biolabs_RestEndoGuide_D1708.pdf
- How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
- Design fragments with compatible ends: The template fragments of interest must have overlapping ends (minimum 18 bp) to allow homologous recombination by exonucleases (generally blunt or overlapping ends).
- Avoid cleavage sites within the regions of interest: If restriction enzymes are to be used to generate the fragments, it is necessary to ensure that they do not cut in areas that alter or compromise the integrity of the fragment.
- Avoid secondary structures: These can interfere with the ligation of the fragment and generate changes in the final structure.