Silly Question time.
wgschech at med.uni-tuebingen.de
Mon Jun 16 04:41:30 EST 1997
I had similar results a few weeks ago, you may find the discussion on
that in the archives here.
As a control experiment, I didn't add ligase to the soup (a cut
plasmid (not dephosphorylated) and an insert (not phosphorylated,
but in excess) and got severak positive colonies with the right
insert. It seems that the annealed but unligated constructs were able
to get into the bacteria and were able to be replicated. Re-ligated
or not. Thats life. Amusing, isn't it? If you have LOTS of your
fragment, you could consider saving on ligase by omitting it.
BTW, what did people do before ligase was invented?
> Given a plasmid digested with a single cutting enzyme, and not
> dephosphorylated, is there an *absolute* requirement for ligase to
> get religation?
> If there is ATP in the buffer but no ligase, I was under the
> impression that such a plasmid would not self-ligate because it
> requires the enzyme to exchange phosphates - at least for a standard
> 6-base cutter. (For longer overlaps it's feasible the cut would be
> 'seen' by the bugs as 2 single-stranded nicks and therefore would
> get repaired in the bacterium).
> Please put my mind at rest ;)
> Richard P. Grant MA DPhil University of Oxford |
> rgrant at molbiol http://www.molbiol.ox.ac.uk/~rgrant FFPGP
> | .ox.ac.uk -----------------------# Trust me - I'm a doctor.
University of Tuebingen
email: wgschech at med.uni-tuebingen.de
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