if we do ligation so we can used 37 centigrade incubator for 4hour or put on ice one hour so which one step is best for plasmid concentration?
According to my experience, incubation at 37 cel for four hours would be the best. This time duration and the temperature allows optimum ligation, especially when if you had dealt with a low concentration of the plasmid. But please refer to manufacturer guidelines. I have also got positive results when I left the reaction mixture in room temperature for 90 minutes.
In addition, after the ligation at the above conditions, you may store the ligated product even in 4 cel until further use.
When doing a ligation you are trying to find a balance between the temperature that optimizes the ends staying together (lower temp is optimal) and enzyme activity (higher temp is optimal). My lab normally did ligations at room temperature for 30-60 minutes. If you do on ice, it will work but 1 hour may not be long enough. Protocols for ligation on ice usually say to leave overnight.
I've usually always done ligations on wet ice (add bit of water to fresh ice) overnight. Make sure to put tubes in a floating holder since obviously will melt by the next day.
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