Week 6 HW: Genetic Circuits Design part I

My Homework

DNA Assembly

  • Answer these questions about the protocol in this week’s lab:

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

Phusion High-Fidelity PCR master mix contains Phusion DNA polymerase, deoxynucleotides and HF reaction buffer with MgCl2. This master mix is used for long or difficult PCR amplifications and applications where high sequence fidelity is critical, such as cloning, mutagenesis, or amplicon sequencing.

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

It is determined by temperature of melting of the primers which depends mainly on the GC content, length of the primer and sequence composition. Salt concentrations and secondary structures could affect primer annealing temperature as well.

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.

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

After PCR, digestion and purification, DNA fragments should be analyzed by agarose gel electrophoresis to confirm the correct size of each fragment. DNA concentration can be measured (e.g., by Nanodrop or Qubit) to ensure proper stoichiometry. It is recommended sequencing the fragment before Gibson assembly to ensure that sequence is free of errors.

5. How does the plasmid DNA enter the E. coli cells during transformation?

It is not necessary to purify Gibson assembly reactions because chemical transformation was performed. Chemical competent cells contain salts such as CaCl2 and MgCl2. Positive ions neutralizes negative charges of DNA and phospholipids in the cell membrane allowing to create a channel during heat shock. Heat-shock creates temporary pores allowing plasmid DNA enter the cells during transformation.

6. Describe another assembly method in detail (such as Golden Gate Assembly)
    1. Explain the other method in 5 - 7 sentences plus diagrams (either handmade or online).
    2. Model this assembly method with Benchling or Asimov Kernel!