Unstable insert

[Rafael Maldonado]

panapat at netscape.net wrote:
> 
> Hi,
>  I have some problem or maintaining large insert in E.coli DH5 alpha.
> The insert size about 10 kbs and vector is pUC18. I got clones that
> contain this recombinant plasmid but the number is very low. Sometimes
> there are some deletion in my gene. What should I do to maintain this
> plasmid in E.coli? I need to get more DNA fro mutagenesis and protein
> expression study.

Besides the recommenddations by Jered, you should use a different
vector. pUC's are not designed to carry those big inserts you are
cloning. A lower copy number vector is more suitable. 

-Rafa 

-- 
Rafael Maldonado
Divison of Genetics
University of Alicante (Spain)