strange transformation results

Peter Cherepanov peter.cherepanov at uz.kuleuven.ac.be
Sat Apr 27 05:00:27 EST 2002


can it be due toxicity of the insert?

For example:
if you prepare your construct by ligating a fragment into a single
restricion site (when both orientations are possible) it is often obvious
then one orientation is much prefferred over another. This happens also with
TA cloning. The effect is further aggravated with IPTG addition (if there is
a lac promoter, for "blue-white" selection) or absence of the lacIq in the
host strain. However, you can most often find the right orientation after
some hunting. (by Murphys law, I do not know the number, may be check here:
http://www.edwards.af.mil/history/docs_html/tidbits/murphy's_law.html the
good orientation is always the rarest one). And usually those ones (at least
to a naked eye) grow as good and form nice same sized colonies.
I explain this effect by higher toxicity of one of the orientations...
can it be a difference in "plating efficiency" because of such toxic effect?

also, if the part of the MCS is still present in the ligation mixture, it
will be religated back. To me it seems that the short DNA fragments ligate
much more efficiently then the long ones (just a kinetic effect, much faster
diffusion?). This will lead to more colonies on the control plate.
Sequencing my constructs I have seen one with the right insert plus three
MCS fragments ligated to each other. Since then I too always purify my
vector on gel.

Peter



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