pBR322 restriction digest - PBR322 digest with EcoRV and BstZ17I (Oct/29/2010 )

Hi,

So after all the hassles of figuring out new restriction enzymes and then the issue with eluting a 15.9Kb band!

I finally have my plasmid which is 15.9kB band and my insert which is 3.3kB and I did a ligation reaction with T4 DNA ligase. And I put in different ratios in a 20uL reaction and left it overnight and transformed and I don't have any colonies!!!!!!!!!!!!

Does anyone have any experience with doing ligations with EcoRV and PacI........I know one of them is a blunt end and the other is a cohesive end......but are there any tricks that I am missing?

I had appreciate the help