Protocol Online logo
Top : Forum Archives: : Molecular Cloning

Fast ligation - (Sep/19/2005 )

Ligation method can not be avoided as I want to clone my insert gene to targeted vector. However, the overnight ligation takes long hour to wait. Is there any way to shorten the ligation time instead of overnight? can it be completed within few minutes? so that the experiment can proceed faster. unsure.gif

thanks..... biggrin.gif


you can use T4 DNA ligase from Roche and ligate at 25 degrees for 4hrs. sometimes 2hrs incubation also works. while working with pGEMT- easy(promega), ligation can be done at 25 degrees for 1 hr.
Some other companies like BG also have instant ligation kit which makes ligation possible in 5 minutes at 25 degrees.I haven't personally tried it so cannot say how efficient it is.
Anyway good luck


I've tried the Rapid Ligation Kitt from Roche and it works pretty good.


I use Takara ligation kit, and it takes only one hour. It's pretty good.


We routinely use Amersham's Ready-to-Go ligase -- done in about an hour.


quikck ligase from biolabs ligate in 5minutes and is good.


All of these are just normal ligase buffers with PEG8000 at a 10-12% concentration. The NEB catalogue gives the original reference.


DNA sequencing

-Daniel Tillett-

I routinely use T4 ligase from NEB and just let it sit on the bench for one hour before transformation. Seems to work just fine.


Use of crowding agents like PEG allow 5-minute sticky end ligations. I use the fast ligase from epicentre. works very well and I like being able to add the ATP separately.