Ligation Problems - (Feb/03/2006 )

Help please
I have done a ligation. I think it worked but I do not know of a way to be sure. I transformed some cells with my ligation product but they are not growing. The vector I ligated my inserts into is Kanamycin resistant. I plated them on Kanamycin plates. The pure vector transformed in comp cells is growing, but my new ligates are not. I am sure I did not cut out the Kanamycin part of the vector. I think I just have a very small amount of ligation product and thus it may be too dilute for the competent cells. Could this be a problem? If so, is there a way I could amplify my ligation products? Is it possible to cut the ligation product (approx. 6kb) Pcr it and re-ligate? Apart from the transformations, how do I properly check that my ligation worked and the concentraion of DNA I actually have. Please help me if you can.

Hey TCells.
I wouldn't worry about having diegested the kanR gene. If you are using a vector with an MCS odds are they won't place a restriction site in there that is in the selection gene. Sounds like your digest is working. If it wasn't you would get transformants with nothing but empty plasmids so it is probably the ligation indeed. Generally you want at least a 3:1 ratio of insert to vector. You can check this spectrophotometrically or by eyeballing how much you have on a gel prior to purifying it (This is what I usually do, I usually don't need to spec it)

What size is the vector?
Also, are you sure you have the insert? 6kb is a lot to PCR, maybe your insert is not correct.
Are you doing the digest straight from the PCR product? If so do you have at least 2 bases next to the restriction site on your oligos?

You shouldn't have to cut out your insert to do PCR on it. You can PCR straight from the whatever vector it is in which it currently resides.

Thanks,
It turns out that it might be a ratio thing. I`m starting over again trying to get a lot more insert. I am also growing cells in order to obtain the vector in higher quantity. Hopefully at the end of it all I will have a good 1:1 ratio at least.

One possibility is that the ligation itself may not have worked. If true, then you are trying to transform bacteria with linearized DNA which does not transform very efficiently at all and will likely produce no colonies. Checking your ligase may be a good idea.

FWIW, I recommend FastLink Ligase from Epicenter Technologies. Excellent ligation efficiency, even in blunt end cloning.

All the best on your next try although I would try at least a 3:1 insert to vector ratio personally. 1:1 probably won't be as efficient if it works.

What is linearized DNA? Why is it hard for it to ligate. My latest ligations still did not work. The positive control is growing well. I`m pulling my hair out. I`m so new at this. Please help. If I do have linearized DNA, is there a way of ligating it. I`m thinking of trying to ligate it into a new vector and then cutting it out from there and putting it in this vector. Does anyone know anything about T/A vectors?
Thanks in advance