Lecture (03/11)
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<img src="/icons/slide_green.svg" alt="/icons/slide_green.svg" width="40px" /> Lecture slides will be shared here after the class!
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<img src="/icons/video-camera_yellow.svg" alt="/icons/video-camera_yellow.svg" width="40px" /> Recording will be updated here after the class!
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Recitation (03/12)
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<img src="/icons/slide_green.svg" alt="/icons/slide_green.svg" width="40px" /> Recitation slides will be shared here after the class!
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<img src="/icons/video-camera_yellow.svg" alt="/icons/video-camera_yellow.svg" width="40px" /> Recording will be updated here after the class!
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Homework
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<img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> These homework questions are based on the Gibson Assembly Lab! Mandatory for both CL and MIT/Harvard students.
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<img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" /> Key Links:
https://docs.google.com/document/d/1_aSV7w8iRYc3EDmbueJ_hSEGy_jHLDfxT2wAezEtC4c/edit?tab=t.0#heading=h.a157u2dx9dhb
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- What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
- What are some factors that determine primer annealing temperature during PCR?
- 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.
- Why does the PvuII digest require CutSmart buffer?
- How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
- How does the plasmid DNA enter the E. coli cells during transformation?
- Describe another assembly method in detail (such as Golden Gate Assembly) 5 - 7 sentences w/ diagrams (either handmade or online). Model this assembly method with Benchling or a similar tool!
1. Components in Phusion High-Fidelity PCR Master Mix and Their Purpose:
- Phusion DNA Polymerase – A high-fidelity enzyme with 3'→5' exonuclease activity, reducing errors during amplification.
- dNTPs (Deoxynucleotide triphosphates) – Provide the building blocks for new DNA strand synthesis.
- MgCl₂ (Magnesium chloride) – A necessary cofactor that stabilizes the enzyme and affects primer binding efficiency.
- Buffer system – Maintains an optimal pH and ionic strength for polymerase activity and DNA denaturation.
- Enhancers/Stabilizers – Improve enzyme performance and ensure robust amplification across GC-rich or complex templates.