Losing pieces of plasmid

[Brett]

>Does anyone have a suggestion that will keep my E. coli from spitting out 
>pieces of plasmid?  I'm trying to put 6kb into a 3kb vector that already 
>has 10kb inserted.  Clones isolated after the ligation don't look like 
>either the parent or the predicted product.  I know that 19kb is pushing 
>it as far as plasmid size, but I'd appreciate any advice.
>
>Thanks
>
>MLK.


It can be done. Use a recA host, of course. Design your ligations to be
very efficient (who doesn't?! ;-)) - no, but don't count on a double blunt
end ligation to jump from 13kb to 19kb. I have had success in 17kb
constructions, and I'm sure you can too. Godd luck!



Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain