What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
Phusion High-Fifelity PCR Master Mix offered by New England Biolabs is a product that contains a DNA polymerase with high fidelity useful for cloning and amplification of difficult amplicons. Phusion DNA polymerase contains proofreading activity (3’ -> 5’ exonuclease) and a higher fidelity 50X greater that Taq polymerase. Thermo Fisher Scientific Phusion High-Fidelity PCR Marter Mix is composed of a HF Buffer or GC Buffer; both buffers are used to reduce the error rate of the DNA polymerase. HF Buffer contains a lower error rate (4.4 x 107) than GC Buffer (9.5 x 107), however GC buffer can improve the performance of the polymerase on some difficult or long templates with high GC-rich templates or with secondary structures. The master mix is also provided with a optimized concentration of MgCl2 which is an essential cofactor that stabilizes the DNA double helix and facilitates primer annealing. High Fidelity PCR reaction can also include DMSO that is used to reduce the melting temperature through its association with Cytosine residues that changes the conformation of the DNA template.
What are some factors that determine primer annealing temperature during PCR?
There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other.
How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
How does the plasmid DNA enter the E. coli cells during transformation?
Describe another assembly method in detail (such as Golden Gate Assembly)
Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).
Model this assembly method with Benchling or Asimov Kernel!