What is the reason for keeping the ligation reaction at 4 degree or 16 degree. I am using Quigen ligation kit. Why do we keep at different temperatures?
Different temperatures are used for blunt end vs sticky end ligations. For blunt end ligations, there is no Watson-Crick base pairing to promote DNA fragment association. This renders the reaction less favorable in thermodynamic terms because there is no enthalpic (bonding energy gain) to offset the entropic cost of fixing the orientation and position of the two DNA fragment required to ligate two DNA fragments. By lowering the temperature (usually 14C, although some folks use 16C or even 4C), you can lower this entropic barrier by decreasing diffusional rates of the DNA.
As Sezer points out, ligase activity is decreased at 4C, so there is a tradeoff between favorable energetics and decreased catalytic rate of the DNA ligase (thus necessitating longer incubations). In practice then, it is a matter of convenience whether one chooses 4C overnight, or a few hours at 14 or 16C.
Different temperatures are used for blunt end vs sticky end ligations. For blunt end ligations, there is no Watson-Crick base pairing to promote DNA fragment association. This renders the reaction less favorable in thermodynamic terms because there is no enthalpic (bonding energy gain) to offset the entropic cost of fixing the orientation and position of the two DNA fragment required to ligate two DNA fragments. By lowering the temperature (usually 14C, although some folks use 16C or even 4C), you can lower this entropic barrier by decreasing diffusional rates of the DNA.
As Sezer points out, ligase activity is decreased at 4C, so there is a tradeoff between favorable energetics and decreased catalytic rate of the DNA ligase (thus necessitating longer incubations). In practice then, it is a matter of convenience whether one chooses 4C overnight, or a few hours at 14 or 16C.
You can do blunt and sticky end ligations at RT in 5 min. at almost the same rate see: https://www.neb.com/products/m2200-quick-ligation-kit. They are using PEG in the buffer to concentrate the DNA ends which increases the rate at which the ligase joins them.
I agree with Daniel Cohen. You can use the thermocycler to get a constant temperature during the ligation reaction
I now do all my ligations at room temperature. It eliminates the need to have different incubation blocks or reserve a thermocycler. They all work.
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