Hi everyone,
I read a lot about the incubation time and temperature of T4 DNA ligases. I'm on a very short time schedule and I was wondering wether or not a 20m at 4°C followed by a 2-3h at 16°C would be sufficient to ligate 25bp insert to a 8298bp vector ? I'm worried that doing a 37°C 1h ligation would be detrimental to such small insert. And I don't have the luxury at the moment to do an overnight ligation.
Should I wait more at 16°C or instead do a RT incubation for a few hours ? (How much ?)
Thanks in advance !
Wilhelm Vaysse--Zinkhöfer
To some degree the answer will depend upon whether you have sticky or blunt ends, and quality of the DNA and the freshness of your ligation buffer. But most of the time I do 30 minute ligations at room temperature and it works fine.
Michael J. Benedik Yeah I should have precise that I've sticky ends, I went for 2h RT and I'll proceed with the transformation in an hour or so. I'll post whatever results I have. Thanks for the answer, that's reassuring, I never tried at RT !
The "optimal" temperature is supposedly 16 where you still have reasonable activity (which increases with temperature) and reasonable annealing (which decreases with temperature). But in my hands RT works perfectly fine.
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