2 replies to this topic

[bekiren]

I'm trying to perfom a simple digestion and ligation reaction to put my insert (~2.4 kb) from the cloning vector to the expression vectors (pET23 and pET28). I get the right size vector cuts and inserts from the big gel and extract them with gel extraction kit and ligate them with T4 ligase, but I don't get any colonies on the plates. I used fresh ligase and ligase buffer, so I don't think this is the problem. We had some contaminating DNA problems in the past, but I am assuming this should be eliminated since the DNAs are gel purified. When we run the gel of the ligation mixtures (after deactivating the ligase), we don't see any band, neither a band for circular DNA or for cut DNA. Does anybody have an idea of what could the problem be? Thanks.

[pDNA]

if you have checked your DNA prior to ligation and see the bands before setting up the ligation reaction then the only thing that could happen during ligation is degradation of your DNA by nucleases.

How long do you incubate the reactions?

What buffer do you use for elution after gel extraction?

Maybe some of your ingredients for the reactions is contaminated with nucleases?

Check if your sample is also degraded within the incubation time without adding insert, vector, ligase, ligase buffer, water and so on.

Regards,
p

[Michaelro]

It might be that the way You estimate DNA concentration after gel extraction is incorrect.
As a consequence, You put very low amount of DNA in the ligation reaction and therefore, You're not able to see it on gel.
I'd recommend to run a small amount of eluted DNA after gel extraction to make sure it works well.
Best
Michael

bekiren, on Jun 8 2010, 08:45 PM, said:

I'm trying to perfom a simple digestion and ligation reaction to put my insert (~2.4 kb) from the cloning vector to the expression vectors (pET23 and pET28). I get the right size vector cuts and inserts from the big gel and extract them with gel extraction kit and ligate them with T4 ligase, but I don't get any colonies on the plates. I used fresh ligase and ligase buffer, so I don't think this is the problem. We had some contaminating DNA problems in the past, but I am assuming this should be eliminated since the DNAs are gel purified. When we run the gel of the ligation mixtures (after deactivating the ligase), we don't see any band, neither a band for circular DNA or for cut DNA. Does anybody have an idea of what could the problem be? Thanks.