why the difference in ligation temperature? - (Sep/25/2007 )
I would like to ask why is ligation being carried out a several different temperature from 4C to 25C? I've tried and found some a lot of different answers online. Can anyone tell me what the optimum temperature for ligation? What is the difference when one does his/her ligation at different temperature and why?
Thank you so much,
For ligation, two things need to happen:
1. The DNA ends need to come together. This involves hydrogen bonding of the complementary bases for cohesive ends or random association for blunt ends. In either case a low temperature such as 4°C will favor this interaction.
2. The ligase has to covalently join the DNA. The optimal temperature (for T4 DNA) ligase activity is 25°C
The temperature normally used is 16°C, which is a compromise for both parts of the reaction. If you ligate at 4°C you will favor part 1 but the ligase will act more slowly so a longer incubation time is needed.