Posted 02 August 2011 - 04:01 AM
I don't know how you have set up your restriction sites. If they are all incompatible sites, then it should be possible to ligate the parts all at the same time. This would be difficult, with many possible things that could go wrong, and with no easy way to debug what is happening, however. I think you would be much better off to ligate and clone shorter fragments, amplifying the amounts of DNA at each stage with minipreps, and allowing sequencing of intermediate results. In the long run, I think this would be easier.
You can visualize ligation products. Use normal (not quick) ligase buffer. Heat kill the ligase. Run a normal gel. A useful debugging technique is to test ligate pairs of parts, which are supposed to ligate, and look for the double length product. You can do this for each pair, and figure out what is ligating and what is not. Your test ligations need not be at low concentrations, although your final circularization reaction should be.