ligation failure-what to do? - tried everything but still not getting transformants (Mar/06/2008 )
plz let me get clear of using TE buffer in ligation.
I've been taught that for restriction digestion, eluting my dna with TE is no need (in our lab we use millipore water which might nat have acidic problem)
but [b]for ligation step i must use TE to elute my dna, which is just the opposite of ur tips (not using TE before ligation).
I won't say i get many colonies, but usually i get my intended vectors.
anyway, it's better to have some more discussions on the point.
plz go ahead.
If you are storing DNA for any length of time, you want it in EDTA to inactivate any nucleases from, e.g, your fingers. In all cases, you want your DNA in a buffered solution with pH around 7.5 or 8.0. The only reason to avoid TE as the buffer you always use is the rare case when you are setting up a reaction where almost all of the volume is DNA in TE, and the 1 mM EDTA in the TE solution affects the concentration of magnesium in the final buffer. If you look at the amounts of magnesium in typical reactions, it is 2-10 mM. For a 2 mM magnesium containing buffered reaction, you MIGHT have a problem if your reaction setup was half DNA in TE -- the magnesium concentration would be down to 1.5 mM. With sane amounts of DNA being added into a reaction, the EDTA content and effect on magnesium concentration is almost negligible. I always elute or redissolve DNA in TE. If, for some reason, I plan on immediately doing a reaction and the DNA is low concentration, I might consider Tris-HCl pH 7.6. I would never use pure water, because of its acidity.
I always make my primers and oligos so that there is at least 5 bases at each end after restriction site regardless of enzyme. It doesnt cost much for extra bases and you dont have worry about not having enough of them.