HTGAA - Week 6: Genetic Circuits Part I: Assembly Technologies

My Homework

Assignment: 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?
2. What are some factors that determine primer annealing temperature during PCR?
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?
5. How does the plasmid DNA enter the E. coli 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!

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Resources

1. Primer Design: HTGAA’s Supplement to Gibson Assembly Recitation
2. NEB’s (New England Biolabs) video Introduction to Gibson Assembly
3. NEB’s (New England Biolabs) explanation & protocols for Gibson Assembly®
4. PCR Master Mix. (s.f.). © Merck. https://www.sigmaaldrich.com/MX/es/technical-documents/technical-article/genomics/pcr/pcr-master-mix
5. PCR Cycling Parameters–Six Key Considerations for Success. (s.f.). © ThermoFisher Scientific. https://www.thermofisher.com/mx/es/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-cycling-considerations.html
6. Restriction Enzyme Digestion. (s.f.). © New England Biolabs. https://www.neb.com/en/applications/cloning-and-synthetic-biology/dna-preparation/restriction-enzyme-digestion
7. 2.14: Restriction Enzyme Digests. (2013). [video]. Basic Biology. JoVE. https://www.jove.com/v/5070/restriction-enzyme-digests-principle-procedure-and-applications
8. Restriction Enzyme Digestion: Key Considerations. (s.f.). © ThermoFisher Scientific. https://www.thermofisher.com/mx/es/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/restriction-enzymes/restriction-enzyme-key-considerations.html
9. PCR Basics. (s.f.). © ThermoFisher Scientific. https://www.thermofisher.com/mx/es/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-basics.html
10. Polymerase Chain Reaction. (s.f.). SnapGene. https://www.snapgene.com/guides/polymerase-chain-reaction
11. Polymerase Chain Reaction (PCR). (s.f.). Addgene. https://www.addgene.org/protocols/pcr/
12. Panja, S., Aich, P., Jana, B., & Basu, T. (2008). How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli? Molecular Membrane Biology, 25(5), 411–422. https://doi.org/10.1080/09687680802187765
13. Bacterial Transformation. (s.f.). Addgene. https://www.addgene.org/protocols/bacterial-transformation/
14. Bacterial Transformation Workflow. (s.f.). © ThermoFisher Scientific. https://www.thermofisher.com/mx/es/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-workflow.html
15. Bacterial transformation & selection. (s.f.). Khan Academy. https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/bacterial-transformation-selection
16. NEBridge® Golden Gate Assembly. (s.f.). © New England Biolabs. https://www.neb.com/en/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly
17. Golden Gate Assembly. (s.f.). SnapGene. https://www.snapgene.com/guides/golden-gate-assembly