RFD: Plasmid re-ligation without ligase?
wgschech at med.uni-tuebingen.de
Mon Apr 21 10:39:36 EST 1997
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
Some additional information on the experiment:
source plasmid structure:
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?
---------!all possible disclaimers apply!-----------
University of Tuebingen
email: wgschech at med.uni-tuebingen.de
public PGP key is avilable on request
More information about the Methods