Golden Gate vs Gibson Yield/Efficiency for Plasmid Construction/Cloning

I needed to prepare a plasmid library and was curious which had the best yield

Bottom line: they are about the same for two component assembly

By gel both rxns are about 25% efficiency
- Both rxns have about 3ng/uL backbone/vector and yield about 0.75ng/uL of plasmid
They gave a similar number of colonies when transformed

Experiment

Both were used to make the same target plasmid
- Both were two component assembly rxns (~3550 bp backbone + ~100bp insert)
- Both rxns had ~3ng/uL backbone
- Loaded 10uL rxn in each well
Gibson – used 20bp overlaps and an
- Used homemade Gibson MM which is amount 2x better than NEB HiFi (both the paper and my independent verification )
- Reference for homemade Gibson: Rabe et al. (2020) A Simple Enhancement for Gibson Isothermal Assembly
- Vector prepared by PCR, insert was 87nt ssDNA, 20bp overlaps
- Used NEB enzymes
- Reacted for 1hr
Golden
- All reactions were run overnight
- cycling: 5 min 37°C → 5 min 16°C for 12 hours
- final 60C 5min heat treatment
- Insert prepared by PCR
- Vector was plasmid with 2 BsaI sites
- Used NEB enzymes/buffer

Conclusions

Gibson and Golden have roughly the same efficiency (compare E with L)
Enhancer (PEG, extra ATP, BSA) makes a big difference on golden gate rxns. I’m not sure which of these components are the important parts.
Golden works equally well with cycling or 22C overnight

Golden Gate details

Gibson Details

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