Silly Question time.

[Wolfgang Schechinger]

Hi Richard!

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?

Cheers, 
Wolfgang



> 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 ;)
> 
> TIA,
> 
> Richard
> 
> --
> 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.
> #-----------------------
> 
> 
-----
                                     
Wolfgang Schechinger         
University of Tuebingen
email: wgschech at med.uni-tuebingen.de

http://www.medizin.uni-tuebingen.de/~wgschech/research.htm

public PGP key is avilable on request