How long for ligations? - (Dec/11/2006 )
I usually incubate my ligations at 16 degrees C overnight. Does it matter how long I leave it? I usually set it up in the afternoon and do the transformation the following morning after about 16hours incubation. Can I leave it for longer? up to 24hours?
I am using T4 ligase from Roche if this helps!
I am using T4 ligase from Roche if this helps!
Hi
I use the same ligase and I don't care when I begin my reaction the day bevor or when I transform the cells the day after. Pay attention that your vector is good digested or CIP inactivated to avoid religation of it, I let overnight the ligation reaction on the bench. In my case it works all the time.
Good luck
I am using T4 ligase from Roche if this helps!
i leave my ligations in RT for 30-60 min. before transforming. it works.
We use Epicenter's Fast-link DNA ligation kit, and it takes 5 minutes at room temperature for ligations.
NEB has a quick ligation kit, where u incubate for 5min at RT.
We use plain ordinary NEB T4 DNA ligase for 10 minutes at room temperature. (For sticky end ligations). We also dilute it 20x. This reminds me of the Fermentas 5 minute "quick enzymes" which are exactly the same as the "slow enzymes" we are all used to using. The wonders of marketing.
I used 16°C overnight and 20°C 2h, there was no difference (with NEB T4 ligase and a Fermentas Kit, where 5 minutes at 22°C was suggested). the 5 min ligation I do not dare to do.
30-60 minutes withT4 DNA ligase, or if I have other work going on, I leave them longer without any problem.
Agree with phage about wonders of marketing, NEB now has "time saver qualified enzymes" which are indeed also exactly the same as the normal ones, just as fermentas, I never have my restriction digests overnight or for more than 1 or 2 hours (only if I have loads of other things going on at the same time I digest longer).
It is not necessary to incubate for a long time. After all you only need one correct colony.
I ever left it over the weekend at RT and it made no different with the one I incubated at RT for about 2 hours. If it works, it will work for the first time. If not, you really have to repeat it for endless time and pray to the cloning god.
Good luck! 
I have rapid DNA ligation kit from ROCHE and it says 5 min at RT. i am doing like that, but currently my ligations are not working. going to test it today if i find some digested and purified vector.