Lab Follow-up

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

Not sure…

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

1. Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp. This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature.

2. Primer Melting Temperature: Primer Melting Temperature (Tm) by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58 oC generally produce the best results. Primers with melting temperatures above 65oC have a tendency for secondary annealing. The GC content of the sequence gives a fair indication of the primer Tm.

3. Primer Annealing Temperature: The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high Ta will produce insufficient primer-template hybridization resulting in low PCR product yield. Too low Ta may possibly lead to non-specific products caused by a high number of base pair mismatches,. Mismatch tolerance is found to have the strongest influence on PCR specificity.

1. 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.

PCR copies fragments of interest by having primers bind to the sequence of interest, creating linear fragments that it copies using temperature cycles. It’s great at amplifying a region of interest from a complex mix of DNA. However you cant use it to turn circular DNA to Linear DNA.

Restriction enzymes create linear DNA by cutting circular DNA at specific sites. It can create clean, predictable cuts on the circular DNA, and is useful if we want to insert/remove specific parts of the circular DNA. However, restrictions enzymes only work when we know the exact sequence in question.

PCR is for specific, small regions of DNA, which must be copied from a larger DNA source. Restriction enzymes are used to create/edit plasmids with known sequences.

1. Why does the PvuII digest require CutSmart buffer?

Enzymes, being proteins, have specific conditions for their optimal activity. Buffers, such as CutSmart, most likely provide an optimal ionic and pH environment for the Pvul to operate in.

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

Gel Electrophoresis. You can also sequence the fragments.