Ligation Difficulty - (May/09/2007 )

My problem is that I have low concentration of three fragments of my DNA after cleaning them with Qiagen purification kit. I want to ligate them together. The two ligation sites have different enzyme cut. I ended up using quite high volume in my ligation reaction (40 ul). It didn't work. Need suggestion on how to make this work.

Thank you.

How much DNA do you have in total and how concentrated is your DNA?

You could certainly try reconcentrating the DNA fragments. That would help witht the ligation. Try ethanol percipitate (with 1/10 volume Sodium Acetate and dextran- if needed)

How if your ligase? The T4 ligase enzyme goes off pretty quickly. Has anybody in the lab recently done a successful ligation or reported problems with their ligation. The ligase buffer also doesn't like freeze thaw cycles. Get some new stuff and remember to aliquote.

For this large 40ul ligation volume... how much ligase enzyme did you add. (should at least be 1ul - 20 U) And how long and at what temperature was the ligation reaction conducted.

I'd say reconcentrating the fragments is your best bet (Assuming your buffer/T4 is okay like perneseblue pointed out) because at a high total volume of ligase reaction and low concentration of DNA the reaction conditions favour recirculasation instead of di- or in this case trimer formation (even if your fragments can't self-ligate they'll still have problems ligating with each other under those conditions)
Precipitate your three fragments in one tube and then directly add your ligation mix (I usually use 10 µl) to the pelleted DNA.

Alternatively (or in addition) you could try adding 5% PEG to the reaction if your buffer doesn't have it already since it works as a volume excluder, making the volume in which the DNA fragments and T4 react with each other smaller, thus favouring inter-molecular reactions.