Ligation of large plasmids

[Bob Kodrzycki]

Anyone have any tips on making large plasmids?  We are having trouble 
constructing a double insert plasmid totaling about 10 kb in size.  The 
first insert of 3 kb goes in without a problem.  However, addition of an 
additional 4 kb hunk of DNA is proving to be very difficult.  I know that 
efficiency of ligation goes down with increasing plasmid size but this is 
a forced cloning with Hind III and Kpn I ends so logically it should work.  
Also for some reason insertion of a double blunt piece is very difficult 
at times.  We are currently using GIBCO/BRL ligase buffer and New England 
Biolabs T4 DNA ligase.  

Any input would be appreciated.

Thanx...Bob K

bkodrzyc at awod.com