20 September 2021 2 7K Report

A lot of answers/literature online say that T4 ligation of >3 fragments is very difficult/occurs at low efficiency.

However, I also see reports/protocols everywhere of Golden Gate being used to assemble dozens and dozens of fragments at high efficiency. There doesn't seem to be that much difference between standard T4 Ligation and Golden Gate except that Golden Gate does the restriction and ligation in one step.

Why is Golden Gate so efficient for 6-24 fragments, but standard T4 ligation is difficult with that many fragments?

My own experiences also suggest Golden Gate is better, but I don't know why:

I tried assembling 4 fragments with standard ligation (after digestion) and got no colonies after several tries, or sometimes some colonies, but the same number of colonies on the "no insert" control plate.

I tried assembling the same 4 fragments with Golden Gate and got ~500 colonies, and only 1 colony on the "no insert" control.

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