How long do you ligate? - (Oct/22/2007 )

Hi,
I've heard people say that they perform their ligations as briefly as 5 minutes and as long as overnight prior to transformation. Any suggestions on ligation time?

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For standard cloning with Ligase 5 U/ul: 5-10 minutes at room tem.
For other cohesive ligation: ligase 1 u/ul, 1h at room tem, if I'm working in the late afternoon or it’s a difficult ligation (because of the size of the fragments) I let it over night at 16 C.
For blunt ends: at 4 C overnight.

Any other???

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generally I ligate overnight at 16 Celsius. An overnight ligation fits with my timetable.

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For all ligations, I use 1ul of NEB ligase

For sticky ends, 15-30 min at RT
blunt ends, 1-2 hrs RT

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Generally I ligate O/N-24 hrs @ 16. This has more to do with my schedule than anything else, by the time I've finished digesting, purifiying, etc its usually late-afternoon/evening. If you have a little more time and plenty of material (dna, ligase, etc) you can set up 2 sets, one ligation at room temp for and hour or 2, and one O/N @ 16. If you get colonies from the RT ligation you've saved a day and can throw out the other one, if not, then transform the O/N ligation.

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Do you ligate ambient or in a thermocycler?

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I have used Rapid DNA Ligation kit from Roche and perform ligations for 15-20 minutes at RT. Has worked for me.

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