Protocol Online logo
Top : Forum Archives: : Molecular Biology

4 versus 16 degree for ligation reaction - (Sep/19/2005 )


there's out there someone that have argument versus 4 c ligation overnight?

i've red so many duifferent opinion about that..

My insert is a DS oligo of 20 bp, my vector is 5 kb double digested with 2 different enzyme...

Luca, ph34r.gif


what do you want to know, I did n't quite get it?


T4 DNA ligase is active in a broad temperature range. The selection of temperature depends on the sticky ends you're using. An AT end will require lower temperature than GC end, for example. You could try putting your ligation reaction in a beaker filled with room temperature water and put it in the fridge to cool overnight.


as recommended by manufacturer's protocol, ligation can be performed overnight at 14 or 16°C. At 4°, ligase activity is strongly reduced...