Henry Wong (hwong at acs.ucalgary.ca) wrote:
> Getting no ligation whatsoever with different ligases from different
> companies (BRL, Promega, Boehringer, Pharmacia), gel purified (Sigma low
> melt), single cut vector religating on itself, blunt (A-overhangs into
> T-vectors) and sticky end ligations,addition of ATP into ligation
> reactions, ligating at 12 degree and 15 as well. What to do, any
> suggestions? Please help. Oh yes, cells are competent.
When you say getting no ligation, are you judging by
transformation alone, or do you look at the ligation rxn on a gel also?
If you are not getting any higher-Mr products on a gel after the rxn,
indicating at least some ligation, I don't have any ideas. But if you see
ligation products on a gel but they don't transform, that was a common
problem for me until I checked the light box I used to look at the low
melt gels when cutting out the bands. Someone had replaced the old UV
bulbs with short-wave, germicidal bulbs. During the long exposure
required to accurately excise the bands I wanted, the short-wave UV was
trashing my DNA. Even if it ligated, it would be so badly damaged as to
be unable to transform. Check your light box; if that's not the problem
hopefully someone else can help.
University of Texas at Dallas