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Lab Follow up

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

Component Application
Phusion DNA Polymerase A high-fidelity DNA polymerase for accurate amplification
dNTPs The building block nucleotides (A, T, C, G) needed for new DNA strand synthesis
MgCl(2) Magnesium is a required cofactor for the polymerase enzyme
Proprietary buffer system (Thermofisher, NEB) These proprietary buffer systems have been optimized to support high-fidelity amplification.

Reference-

  1. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012771_Phusion_HiFi_PCR_MasterMix_100rxn_UG.pdf
  2. https://www.neb.com/en-us/products/m0531-phusion-high-fidelity-pcr-master-mix-with-hf-buffer

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

Factor How does it affect the annealing temperature?
Primer length Longer primers have higher annealing temps
Primer GC content Higher GC% increases annealing temp (3 H-bonds vs 2)
Complementarity to template Better match allows lower annealing temp
Salt concentration Higher salt raises annealing temp by stabilizing dsDNA

Reference-

  1. Obradovic J, Jurisic V, Tosic N, Mrdjanovic J, Perin B, Pavlovic S, Djordjevic N. Optimization of PCR conditions for amplification of GC-Rich EGFR promoter sequence. J Clin Lab Anal. 2013 Nov 27(6):487-93. doi: 10.1002/jcla.21632. PMID: 24218132; PMCID: PMC6807403.

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.

The protocol uses PCR to amplify two DNA inserts and restriction digest with PvuII to linearize the pUC19 plasmid.

PCR amplification Restriction enzyme digest
Uses a DNA polymerase to amplify a specific region of a DNA template. Uses primers to define the ends of the amplified sequence. Uses enzymes that cut DNA at specific recognition sequences. Uses PvuII enzyme to cut pUC19 at defined sites
Can add sequences (overlap regions) not present in the original template. Limited to sequences already flanked by appropriate restriction sites. Limited to sequences flanked by PvuII sites.
Better for extracting a specific region from a larger DNA Appropriate for linearizing a plasmid. Simpler protocol (mix components and incubate)

Why does the PvuII digest require CutSmart buffer?

CutSmart is needed for optimal activity of the PvuII enzyme. It provides the proper pH, salt concentrations, and cofactors for efficient cleavage at the PvuII recognition sites. (Restriction enzyme digestion)

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

To ensure the digested plasmid and PCR inserts are suitable for Gibson assembly: