Cloning problem

Sergio add at yahoo.com
Mon Nov 11 08:05:45 EST 2002



E Patterson wrote:
> If you're using just a a run of the mill plasmid, my first guess is
> you're REALLY not supposed to try to stick a 14kb insert into a 4kb
> plasmid.  From what I recall plasmids generally can only handle <7kb. 
> Read the plasmids manual and it will probably say in the intro what the
> maximum size is.  You'll probably need a cosmid for something that's
> 14kb.  I've not ever worked with them before though because I've never
> stuck more than 2kb into a plasmid.
> 

We have been able to clone more than 16 kb in a pUC (yielding a >19kb plasmid), 
and the plasmid is working ok (no replication or recombiantion problems). We 
have got even bigger plasmids with different replication origins.
I don't like the use of SAP since (in my hands) the ligation efficiency is much 
lower. Since the plasmid ends are not compatible, I would try the experiment 
with a big amount of insert and non-dephosphorylated vector.
Have you consider the possibility of toxic effects of the proteins encoded in 
the insert?
Is the plasmid you are using an expression vector? In that case, avoid the use 
of inducer until you get the correct clone.


Sergio




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