Ligation

[Dott. Berrino]

I need to ligate toghether two single fragments of dsDNA obtained from two digestion reactions with the same restriction endonuclease whihc generates sticky ends. I also need to obtain a clear band on an agarose gel for my cloning strategy. I've tried several times with T4 DNA ligase but I couldn't obtain the desired band. (I obtain no band or a smear)
Is there anyone who can help me?

[Jelanen]

Two dumb questions right off the top of my head(pointy):

Are you absolutely sure that you are only getting one cut in each strand? Are you sure that your restriction enzyme is fresh/active and that its able to cut what you want?

I know they are dumb questions, but I've found its usually the little stuff like this that gets missed...

-Jelanen

[Dott. Berrino]

Absolutely not dumb questions, I'm sure the enzyme cuts just once (I've choosen it on porpuse!), and I’m sure it cuts ‘cause I visualised it on a gel! But the Problem seems to be Ligation! Where is DNA band after ligation???
I’ ve also tried to amplify the ligation product but I obtained just a smear!

[Jelanen]

Ok, to troubleshoot this problem better I need some more info... at what voltage are you running the gel and for how long? What concentration of agarose are you using? Are you absolutely, positively sure that your stuff isn't contaminated because you saying that you are getting a smear sounds more like you are making DNA puree instead of a ligation. Make sure the stuff you are using says something about being tested for DNAase.
-Jelanen

[Dott. Berrino]

voltage: 120 volt for approximately 40 min.
% agarose:I tried many % from 0.6%to 1% agarose in TBE
I can suppose that DNA has not been contaminated cause on tha same gel I can visualize clear bands of fragments after digestion. The same fragments after ligation and amplification show the bad smear. The only thing I'm adding after cutting is the enzyme (just bought and purity certified).I know that ligation gives problems when visualized on a gel but after amplification I'm expecting a band of a good quality.