Problems with ligation after GeneCleaning
houchens at med.uvm.edu
Sun Aug 25 08:34:37 EST 1996
Anis Limami (limami at versailles.inra.fr) wrote:
: I suffered with the same probleme as you , it was impossible
: to get ligation succesfull with fragments purified by
: GeanClean after enzymic digestion, I dont no why.
: The only thing worked at the end in my hands is the "old"
: purification by electroelution followed by phenol-chloroform
: purification and alcohol precipitation.
I experienced the very same problem as all of you, although the cause of
my woes may have differed from others. Somehow, somebody contaminated our
GeneClean beads with the same vector (supercoiled) plasmid that I was
trying to clone into. So my ligation controls were fine (no insert, no
contamination, no transformants), and the more insert I would ligate with
(i.e. the more contamination that I was adding) - the greater number of
transformants I would get back. But when I minipreped my transformants,
all I got back was the vector w/o insert. I finally took some of the
beads (no DNA), washed them a few times with New Wash, and eluted in water
at 55 degrees (standard procedure for fragment purification) and
transformed cells. Guess what? The more I added, the more greater number
of transformants I got. So, although this may not be the reason for the
problems that your are experiencing, it is always wise to suspect that
the reagents that you are working with are not entirely clean - esp. if
they are used by other members of your lab.
(houchens at salus.med.uvm.edu)
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