Week 6 Lab: Gibson Assembly

Gibson Assembly Lab

Pre-Lab: Primer and PCR (Part 1 of 3)

  • Read this section and scanned the NuPack software hyperlink

Pre-Lab: Gibson Assembly (Part 2 of 3)

  • Read this section

Pre-Lab: DpnI

  • Read this section

Pre-Lab: Plasmid Transformation

  • Read this section

Part 1: Polymerase Chain Reaction (PCR)

Prepared PCR (see photos below)

GibsonLab_1 GibsonLab_1GibsonLab_2 GibsonLab_2GibsonLab_3 GibsonLab_3GibsonLab_4 GibsonLab_4GibsonLab_5 GibsonLab_5GibsonLab_6 GibsonLab_6GibsonLab_7 GibsonLab_7GibsonLab_8 GibsonLab_8GibsonLab_9 GibsonLab_9GibsonLab_10 GibsonLab_10

Part 1a: DpnI Digest

Completed DpnI Digest (see photos below)

GibsonLab_11 GibsonLab_11GibsonLab_12 GibsonLab_12

Part 1b: DNA Purification and Quantification

Purified and quantified DNA. It seems at this point that I did something wrong in one of the proceeding protocol stages with my non-Blue chosen color, so instead of proceeding with both colors, I only proceeded with Blue, as the other color did not have an adequate concentration. See photos below for more documentation of this protocol step

GibsonLab_13 GibsonLab_13GibsonLab_14 GibsonLab_14GibsonLab_15 GibsonLab_15GibsonLab_16 GibsonLab_16GibsonLab_17 GibsonLab_17

Part 2a: Gibson Assembly

Completed Gibson Assembly. Incubated reaction per protocol (see photo below)

GibsonLab_18 GibsonLab_18

Part 2b: Transformation

Completed Transformation protocol step

GibsonLab_19 GibsonLab_19GibsonLab_20 GibsonLab_20GibsonLab_21 GibsonLab_21

Final Results

LOREM

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Supporting prompts for analyzing the lab protocol listed below for reference

Supporting PromptModel
There’s a part of this page that says “After PCR, we treat each reaction with DpnI to eliminate carryover of the original mUAV plasmid.”. That’s Dpnl, not DpnI right? Is it a lower-case l or a capital I?Gemini 2.5 Flash
Remind me what an oligonucleotide is again in simple terms. Keep the response to this prompt short and do NOT hallucinate/make anything upGemini 2.5 Flash
Within the context of this lab, what is a ‘HiFi assembly method’? What is an ‘overhang’? Keep the response to this prompt short and do NOT hallucinate/make anything upGemini 2.5 Flash
How does the ’exonuclease “chews back” one strand of the double-stranded DNA.Gemini 2.5 Flash
I guess I want to understand how this exonuclease works at the chemical level in relatively simple terms. Can you explain that for me? Do NOT hallucinate/make anything up when replying to this promptGemini 2.5 Flash
When the lab refers to ‘Secondary Structures’, what does that mean? Explain in relatively simple terms and do NOT hallucinate/make things upGemini 2.5 Flash
Within the context of this lab, tell me what the ‘pUC19 backbone’ is? What does ‘pUC19’ stand for? Answer this prompt in relatively simple terms, keep the response relatively short, and do NOT hallucinate/make things upGemini 2.5 Flash
Believe when this lab refers to ‘molar ratios’ it means the ratio of molecules to one another. Is this correct or am I mistaken? Do NOT hallucinate when replying to this prompt and answer this prompt in relatively simple termsGemini 2.5 Flash
When the lab states, ‘DpnI recognizes the sequence GATC only when it is methylated’, the ‘GATC’ refers to a Guanine-Adenine-Thyme-Cytosine combination/piece of DNA, and methylation is a chemical process related to said combination/piece of DNA, correct? Do we refer to a 4-letter combination/piece of DNA as a codon, or am I mistaken? Explain in simple terms what methylation is, the actually technical name of this 4-letter combination/piece of DNA, and if there was anything about my thinking/statements so far that’s off. Do NOT hallucinate/make things up when replying to this prompt and keep things relatively simple and short whenever possibleGemini 2.5 Flash
What does the ‘SOC’ in ‘SOC growth media’ stand for? Do NOT hallucinate/make things up when replying to this prompt and answer this prompt in relatively simple termsGemini 2.5 Flash
In the context of this lab, what does ‘uM’ stand for/mean? What does ‘uL’ stand for/mean? Which represents a larger volume? Explain this in relatively simple terms, show me where these terms fit on an overall scale of liqiuid volumes, and do NOT hallucinate/make things up when answering this promptGemini 2.5 Flash
What is ‘silica adsorption’? Do NOT hallucinate/make things up when answering this prompt and keep the answer relatively conciseGemini 2.5 Flash
When the protocol says to ‘gel at ~ 100 mV for 15 min.’, the ‘mV’ being referred to is some type of voltage, correct? Do NOT hallucinate/make things up when answering this promptGemini 2.5 Flash
How does Chloramphenicol relate to amilCP? Are they the same? Clarify this relationship and do so in relatively simple terms and do NOT hallucinate/make things up when doing soGemini 2.5 Flash