Deleted plasmids often result from re-ligation of your DNA in bacteria.
The fact that you see them by itself means that your cells are of quite
a good competency.
before you conclude that the insert is toxic:
1) check your ligation mixes on gel - if there was any degradation
(downward smearing) just re-prep your fragments.
2) do your ligations overnight, 16C (many say 1 hour "always" works,
however, longer ligation does always improve the odds).
3) check more colonies, of course. If you have a pair of primers (one in
the vector and one in the insert) it is easiest to check many by PCR
(directly from colonies).
4) Finally: avoid long UV exposure (!) with longer fragments it is even
more crucial. It can "kill" your DNA, and you even will not know it. If
you need more than 3 seconds to cut the band, it is too long (hand-held
low power UV lamp is the best). If you use a transilluminator for that
(it is a killer), be very carefull and illuminate only the part of the
gel that you are working with (one band at a time) and WORK FAST.
[Only registered users see links. ] wrote:
It is weeks that I am trying to obtain a good contruction, I first did
ligations with these agarose
gel purified fragments, then I set up different ligation with different
molecular ratio 3:1, 1:1, 1:3
and I transfected E. Coli. The transfection was not very efficient but I
obtain several nice colonies
with all ratios and the control (cutted fosmid alone) was good (no
But once I do minipreps and digestion controls, my construct is much
more shorter than what I
expect and not allways the same (8kb to 25kb). I think that there is
some deletions that occur
within bacteria and I tried different bacteria (DH5alpha heatshock, DH10
heatshock) but I always have the same problem. Is my insert toxic? Do
you know some bacteria
strain wich is more appropriate fore such huge construct?
I would be very grateful if someone could give me some goog advices!