RFD: Plasmid re-ligation without ligase?

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Mon Apr 21 10:39:36 EST 1997


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?

Some additional information on the experiment:

source plasmid structure:

>-------EcoRI-(400)-ApaI-(800)-EcoRI-ApaI------->

digested with ApaI to yield a fragment of 800 bp and the vector. The 
vector was re-ligated.

I examined the minipreps by doing a double digest with EcoRI and 
ApaI. This yielded one fragment of 400 bp and the vector.

What do you think: Have I twisted my brain (if present at all), is 
this result normal or what?

Wolfgang




---------!all possible disclaimers apply!-----------
                                     
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	



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