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Why ligation reactions are done at 4C - Ligation of insert into vector (Apr/04/2005 )

Could anyone tell me the reason as to why ligation reactions are performed at 4 deg C? Why, that specific temperature??



the temperature of the ligation reaction is not always 4°C. It depends on the enzyme, on your buffer settings etc... .
You can also ligate insert and vector in 30 min. at 14-16°C, or overnight at 4°C...

It has probably got something to do to minimize unspecific reactions of the "T4 Ligase". The more stingent you are with the temperature the more unspecific reactions are avoided.
And maybe, keeping linearized dsDNA at room temperature might result in degradation by maybe other enzym contaminations, which are not active a 4°C.

Well, I´m only guessing; anyway maybe it´s some truth behind it.


you can do a 'fast ligation' at room temp for an hour.

i do overnight at 16C.