Melany Jumbo, Quito, Ecuador

Genetic Circuits Part I

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

DNA Pol (Phusion) = This Polymerase is better than Taq Polymerase. We can use this DNA Pol because it has a error rate 50 times lower than Taq Pol.

Buffer (HF with MgCl2) = This one optimize de reaction conditions. It gives more enzimatic stability. Also gives to Phusion DNA Pol the Mg+ that it needs to works better.

dNTPs = This are all the nucleotides that are the basic blocks to sintetize DNA.

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

GC Content = Guanine and Citocine that have triple bonds, which elevates the alignment temprature

Primer length = Longer primers requires high temperatures.

Melting Temperature = Tm=4(G+C)+2(A+T).  3 to 5 Grades Celcius under Tm primer.

  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 Enzyme digest
Uses Modification and Amplification of the DNA Generates compatible ends (ex. Clonation)
Advantajes We don’t need pre-existing sequences. We can have a high precisions cuts.
Limitations Amplification errors. Limited to specific sites defined by the available enzymes
Specificity Depends on the primer design Depends on the restriction site.
Mechanism Amplify specific fragments using primers and polymerase. Cuts DNA at specific sites recognized by enzymes
  1. Why does the PvuII digest require CutSmart buffer?

CutSmart Buffer provides optimal conditions (pH, salts, BSA) for PvuII to function efficiently. This buffer is also compatible with many other enzymes, simplifying protocols requiring multiple digestions.