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Background

I've always been told that DNA assembly can be tricky when using very small DNA parts due to low efficiency. I've also seen this when using 3A biobrick assembly to assemble promoters and RBSs together. However, there doesn't seem to be any information that I can find which definitively shows what the minimum DNA part size is that can be used before the efficiency becomes so low that assembly is unfeasible. The most I can find are warnings that small DNA parts are problematic (e.g. https://blog.addgene.org/plasmids-101-golden-gate-cloning).

The Question

What is the minimum DNA part size that can be used in DNA assembly before it becomes unfeasible? I'm specifically interested in Golden Gate assembly, but information for other digest-ligate assembly methods could also be helpful.

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Purely going off experience here having used golden gate assembly methods for 5+ years now, there is a definite lack of literature regarding small part assemblies. In my current lab, we use CIDAR MoClo (a golden gate method) and have assembled 100's of constructs successfully with parts as small as ~ 70 bp. However, when we assemble smaller parts we tend to increase the amount of this part compared to bigger parts. For example, we dilute each part to 10 fmol ready for assembly as a basis for all assemblies, this then allows us to play with the ratios and quickly optimise reactions. So as a standard, when the part is small we automatically start with at least a 3:1 instead of 1:1, where we put 3x the volume of the small part compared to others in the assembly. This usually helps, but on top of that we try to increase transformation efficiency by using SOC media and generally avoid building parts < 100 bp. e.g. we don't tend to use promoters and RBS separately we make larger combined parts containing both.

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I don't have hard data to share regarding you question, but I can provide some anecdotal evidence. I have used golden gate assembly to build hundreds of plasmids using ~20bp annealed oligos, as well as hundreds of plasmids using ~70bp promoter+RBS, 720 bp eGFP, and ~70 bp terminators encoded on plasmids into a ~6kb backbone. In these cases I use 40 fmol backbone (1:3 backbone to insert molar ratio), and I typically get 50-100 colonies. In my experience it is the number of fragments more so than the minimum size of the fragments that has reduced efficiency. I have, however, seen reduced efficency with larger inserts. For insertion of 15-20 kb insertions into a ~6kb backbone I see far fewer colonies, however I haven't teased out if this is lower reaction efficiency, or (what I expect to be the case) reduced transformation efficiency.

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It may also be worth considering how you are amplifying and isolating your small part. Gel extraction kits sometimes have an optimum DNA size ranges that may prevent recovery. Or if your part is similar to your primer length you may see considerable contamination in your 'purified' product if say the size of a single RBS.

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