I need help on Ligations

[George Theodoris]

Andy Kravetz (akravetz at MO.NET) wrote:
: why. Here are some facts and maybe y'all can help me. The insert was PCR 
: amplified to have the two restriction sites that I need, Nco1 and EcoR1. 

As the other people who responded have mentioned, make sure the 
restriction sites are far enough away from the ends of the fragment to 
get good cutting.

: water bath that can hold 16C in the cold room. The conc. of the two are 
: Insert 90ng/ul and Vector 500ng/ul. 

I think your concentration of insert is kind of low. I would start with  
more insert. Since you are PCR amplifying the insert it shouldn't be 
hard to get more of it. When I have trouble cloning things I always 
increase the amount of insert I use in the ligation. That always 
works. Sometimes you just have to bash the dam things in there.
Hope this helps,
George