strange cloning result
wgallin at gpu.srv.ualberta.ca
Mon Mar 27 17:14:37 EST 2000
Wolfgang Schechinger wrote:
> Hi all,
> recently, I performed a simple cloning experiment (sticky
> HindIII-XbaI insert into pCDNA3).
> As directed in the textbooks, I had an control ligation without the
> insert. This control gave me about 300 colonies as "background". The
> sample containing the insert yielded 100 colonies only of which I
> prepped 24. Finally 15 (60%) contained the insert though I didn't
> expect to get any positive. Is there any reasonable explanation for
> this except statistics? Was it an insert overdose killing most bugs
> due to too long insert concatenations? (Concentrations were estimated
> on gel, insert to host ratio was 5)
I see this fairly regularly (which probably doesn't speak too well
for my cloning technique, as you will see).
The explanation that I finally came up with is that the double
digest of the vector is not working as well as expected. Thus, you have
a small amount of simply linearized plasmid, which re-ligates like crazy
and gives you the high background. In the presence of the insert a
significant portion of the simply linearized vector ligates to the
double-cut insert, yielding a recombinant linear molecule that can not
circularize because the remaining ends are not compatible. This leads
to a decrease in the background clones and the appearance of the
expected recombinant plasmids occurring at the same time; sometimes the
decrease in the background exceeds the increase in desired recombinants.
Anyone have a different explanation?
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