Hi Shridhar Kinkar

With NEB T4 DNA ligase (not the fast one), I incubate the ligation mixture for 30min-1h at room temperature and do the transformation. Normally I use 1 vector: 5 insert for my ligations and it works perfectly fine.

Cheers

Hi Shridhar Kinkar

With NEB T4 DNA ligase (not the fast one), I incubate the ligation mixture for 30min-1h at room temperature and do the transformation. Normally I use 1 vector: 5 insert for my ligations and it works perfectly fine.

Cheers

It depends a lot if you have cohesives ends or if they are recessive or blunt-ends

Jean-Pierre Dangy I have insert/vector with cohesive ends.

So it will probably be done in a short time of incubation at R.T

I know that one of the differences between the quick ligases and standard T4 is the inclusion of PEG in their buffer as a crowding agent. You could try adding some PEG 8000 to your mix? I'v never tried it with T4, but it might work. It could impact on transformations though, if you add too much.

I have zero experience with T4s other than the Thermo Scientific's T4 DNA

Ligase (Cat #EL0016). The recommended time of incubation is only 10 minutes (at 22 degree C) for it. If the protocol for the enzyme which you are using says 16h is optimum then I think its better to incubate for 16h.

You can use 10 min, if this time is recomended or you can use more time if the ligation efficiency is low at 10 min. The best result of ligation is relared with the proportions of insert:plasmid and how do you clean the DNA. I usually use no more than 100 ng (totally 1:3 plasmid:insert) and I clean the DNA from gel with any cleanning kit. I hope that this information coud be useful.