Blunt-Cohesive ends ligation - Blunt-Cohesive ends ligation (Jul/06/2005 )

Hi,
I have an insert cloned into TOPO vector and I want to interrupt this insert with an antibiotic resistance gene.
I cut the insert cloned in TOPO vector with NruI and NheI. The restriction resulted in 5kbp fragment and a small 700bp fragment. I also cut the vector that has the resistance marker gene with EcoRV and XbaI. The restriction resulted in two closed fragments around 3kbp, the fragment I need is the largest.
I purified the TOPO/insert vector and the 3kbp fragment and I set up a ligation reaction. The insert (around 3kbp) : vector (around 5kbp) molar ratio was 5:1, and the ligation reaction was 16 hours 15ºC.
This reaction is a blunt (NruI-EcoRV) – cohesive (NheI-XbaI, compatible ends) one.
When I saw my ligation product in agarose gel, I observed new bands that seem to be that the ligation reaction worked. However when I transformed it in TOP10 F’ competent cells, I had no recombinants.
The results have been always the same no matter what I change.
I set up a control reaction, digesting the resistance gene/vector and re-ligating it, and in this case I did obtained recombinants, only if I did not purified the fragments digested. When I purified them, I didn’t obtained recombinants. So, it seems to be a purification problem, but I also think that is a ligation problem, and observing the bands in the agarose gel after ligation, I think that only one end is ligating. I have changed the band purification system using resin kits and electroelution.
Do you think that a blunt-cohesive ligation reaction is too difficult to perform? What ligation conditions do you think I should use in this case (I’m using Invitrogen ligase, and I’m following the blunt conditions they recommend).

Thanks a lot for any suggestion

MM

Hi MM20,

Sorry I got a little lost in your explanation. Your 5 to 1 molar ratio was this calculated after gel purification or from the initial starting amount digested? I firmly believe that gel purification methods are not efficient unless there is ample DNA to start with. What I would want to do (and you may have already done this) is visualise the DNA on a gel after purification and before ligation from this visualisation I would then set up a number of ratios ranging from 1 to 1, 5 to 1 and 10 to 1 (insert to vector). I would also try to keep the concentrations on the higher end of the spectrum.

Hope this helps,

Scott

Hi Scott,
The molar ratio was calculated after gel purification, just by visualising the DNA on a gel. I’ll try with other ratios.
Thanks a lot
MM