What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
The mix contains the required elements for a PCR, namely the polymerase, Mg ions, a stabilising buffer, and deoxynucleotides in equal amounts.
The polymerase, a thermostable enzyme from the Pyrococcus archeobacteria, is in charge of the reaction itself, attaching at the end of the primer/template complex and incorporating new complementary dNTPs in order to extend the chain. Mg ions are present to increase stability of the primer/template, while the buffer supplements other ions for optimal enzymatic activity.
What are some factors that determine primer annealing temperature during PCR?
The complexity of a PCR reaction can vary depending on the type strategy, the size, or the elements to be amplified. However, there are several elements that remain constant in regards to troubleshooting. Annealing, for example, can be affected by elements such as the difference between melting temperature (Tm) of primers and the template. The larger that difference is the less efficient the annealing is. But that is the more basic one.
Today, with PCR mastermixes being pre-made, other elements such as ion concentration (Mg, Na, K), can be much more controlled and standardised. Furthermore, the quality of the DNA template and the amount of base complementarity would be big elements to take into account. The more the primer fully complements the template, the better, and it is also reccomended to have a higher GC content (keeping the Tm range in mind), ideally including two G/C bindings at the 3’ end to ensure a proper binding. The presence of alternative nucleotides can also affect the reaction, although this is not something present in every case.
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 main aim of each of those techniques is very different. Restriction digest essentially is used for cuts in sequences of interest, normally to either obtain a sequence of interest that can then be used downstream, or as an identifier, producing a known band pattern to indicate the presence of a particular DNA template in the sample. They could be of use in cloning, to determine whether or not a particular sequence has been integrated in a plasmid or not.
PCR, while also a technique that allows us to identify the presence of a particular sequence in a sample, has the capacity to replicate the sequence of interest, allowing us to increase the concentration for downstream uses, such as cloning strategies, or even restriction digests. The technique is useful in assembling novel sequences from an original template in order to design new genetic structures.
In the end, bothe techniques are part of the same arsenal of procedures that allow us to developing proper cloning strategies from primer design to gel identification.
Why does the PvuII digest require CutSmart buffer?
The reaction requires CutSmart buffer because this particular buffer has been designed to optimise enzyme activity, as seen in the NEB website.
How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
The best way to do this is to design the primers appropriately. Either designing them with an overhang that would allow the resulting sequence to have extra nucleotides that would themselves be complementary to the contiguous Gibson fragment, or designing every primer to be used for Gibson with complementary cutting sites (Which would have to be different for every fragment, in order to ensure each of them binds only in the desired position)
How does the plasmid DNA enter the E. coli cells during transformation?
In order for cells to be more susceptible for DNA transformation, they need to be prepared accordingly and modified for such a procedure. They can either be electrically or chemically competent but they are, in the end, simply made more porous. In the first case, the electrical shock creates slight dissassembly of the bacterial membrane, with pores by which DNA can enter. The Chemically competent cells work via a similar process, only triggered by a temperature shock.
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 cloning was first introduced in https://pubmed.ncbi.nlm.nih.gov/18985154/.
Starting from the desired plasmid backbone, primers are designed, containing an overhang (a sequence not present in the original template) which introduces a BsaI/Eco31I cutting site.
Compatible ends are included into the tails of the primers used to amplify the fragment to be introduced in the backbone.
Given how Eco31I cuts, after re-ligation, the original cutting sites disappear, making the DNA insertion seamless.
As an example, here is some of my own work:
