Week 6 HW: Genetic Circuits Part I
Assignment: DNA Assembly
1. What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
From New England Biolabs “Phusion High-Fidelity PCR Master Mix with HF Buffer is a 2X master mix consisting of Phusion DNA Polymerase, deoxynucleotides and reaction buffer that has been optimized and includes MgCl2. All that is required is the addition of template, primers and water.”
2. What are some factors that determine primer annealing temperature during PCR?
From ThermoFisher Scientific “The annealing temperature is determined by calculating the melting temperature (Tm) of the selected primers for PCR amplification. A general rule of thumb is to begin with an annealing temperature 3–5°C lower than the lowest Tm of the primers … One important consideration in Tm calculation is the use of PCR additives, co-solvents, and modified nucleotides. The presence of these reagents lowers the Tm of the primer-template complex.”
3. 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.
PCR is used to make many copies of a section of DNA. Restriction enzyme digests cut the DNA at specific points, and do not make multiple copies.