RFD: Plasmid re-ligation without ligase?

Klaus Salger salger at wap1.zi.biologie.uni-muenchen.de
Mon Apr 21 13:38:56 EST 1997


Wolfgang Schechinger (wgschech at med.uni-tuebingen.de) wrote:
: Hi!

: Just had a result I wanted to place here for discussion and comments:

: I cut a plasmid into two fragments, purified them on an agarose gel 
: and religated one fragment. Worked as it should. I got the plasmid 
: with the desired deletion.

: I ran a control without ligase and got colonies there (estimated 
: 1/100 compared to the experiment with ligase) after transfection od 
: XL1-blue. I tested two of these, randomly picked, for the plasmid: 
: Interestingly they seem to carry the re-ligated plasmid. At least, 
: the isolated plasmids don't carry the fragment I had cut out and 
: appear to be identically to the ligated plasmids after a digest. 
: Is it possible that a sticky ligation just can be driven by a 
: chemical equilibrium? And that in an amount that make ligase resemble 
: to be unnecessary? I wouldn't expect that unligated plasmid DNA is 
: stable in E. coli. It should be degraded by endonucleases quite fast. 
: Might E. coli's DNA polymerase be able to read over sticky 
: non-ligated DNA?
<snip>


Wolfgang,

the colis got there own ligase so I would expect a certain fraction of the
linearized plasmids to be religated in vivo and propagated as usual rather
than being degraded (by exonucleases?).

Cheers
  Klaus

--
Klaus Salger		phone : +49 (0)89 5902 	-502
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