Ligation at 16°C - problems wit ligation (Sep/21/2005 )

Hi

I have been having some problems with some ligations I ve been doing. My vector are about 5 Kb and my inserts 300 bp, 600 bp and 800 bp (there are 3 diferent ligations) all cutted with 2 diferent enzymes that leaves stiky ends. I am using T4 ligase from biolabs.

In their protocol the people of biolabs recomend to do the reacctions at room temperature for 10 minutes but in the lab were I am now people has proven to do left the reactions at 16°C overnight or atleast 1 hour, It seems to work for them.

I would like to know wich temperature or time could be better.

If somebody can help me I will really apreciate it

Tanks

There is another thread with this same topic. Some new "fast" ligation reagents claim 5minutes at room temp. for a double sticky end ligation. This has worked for me many times with the fast liagase from epicentre. Some of the liagases without crowding agents still claim you need 16 degrees for 1 hour. Check the protocol for the company you bought the enzyme from.

I usally do my ligation at 4 degree overnight (DIRECTLY in the fridge) and have always worked ( at least for sticky end..)
At this temp you have a good compromise between activity of ligase and selectivity, more the temp is high, more problem you could have..


Luca,

All of my ligations sticky/blunt single double have worked ine at 16C o/n. Sometimes I even keep or 24 hrs at 16c during weekends

We use the Roche Rapid DNA Ligation kit that claims 5 min at RT (though I tend to let it go 15-20 min for good measure). I have also used regular T4 ligase and buffer and ligated O/N at 16oC. In the last ligation I did, I found that the O/N at 16oC worked a bit better (less additional ligation products), but the Rapid Kit is great when you want to do the ligation and transformation in one day.

Save the time and have faster results. I don't understand why people still use these o/n ligations while these 5 minute protocols are now well established.