thanx again everyone!

Aimikins, the kit paper says: concerning the lenght of incubation (5-30min at 16C), it does not matter what kind of ends the DNA fragment has. what do you think?

I use the high concentration ligase from Invitrogen: it also says to use it for 5 min. In the past I didn't trust it and I ligated for at least 20 min but I realized that the ligase is more efficient if you do it for 5 min. I don't really know why (I could guess) but it works better in 5 min.

Relax, Kathy -- Forget what the marketing gurus at the kit compaies say (5-minute ligation! Twice as fast as Competitor X!), and think about what the components of the kit are doing -- you cannot over ligate (but you can under ligate).

Give yourself the best chance of success by using a bit more ligase, a longer ligation time, and an incubation temperature of 16°C...

kathy, u could set up a couple of ligations and try a time course of ligations kept for dif. time points.

U will use a bit more of the ligase but u should have a product and will know the next time which conditions r best suited for u.

dnafactory -- it was coincidental that my post followed yours; I wasn't implying that your were an Invitrogen sales rep...

It just seemed to me that Kathy was getting a little hung up on her ligation kit directions, and I wanted to suggest that she should look past the instructions (which are optimized, sometimes, to allow marketing people to claim they're "twice as fast as Competitor X" and other such nonsense) and think about what the components of the kit are doing...

I'm with HB on this one

kathy, I would still ligate longer. sometimes the reaction just isn't efficient without tweaking it around a little

thanx a lot everyone! i will try ordinary 30 min ligation and overnight ligation and see which works better. will keep you posted.