At the moment I need to clone big inserts in an expression vector (pcDNA3.1+). The vector size is 5.4 kb, and my inserts range from 5.1 to 4.2 kb, which is already difficult, but I also have to cut with different enzymes. The pcDNA3.1+ vector is cut with EcoRI ans AscI (sequential digest, starting with EcoRI), and the inserts are cut with MfeI and MluI (also sequential digest, starting with MfeI). I've already used a 1:1, a 1:3, and a 1:5 vector:insert ratio, using 20 or 30 ng of pcDNA3.1+. I use T4 DNA ligase (high concentration), and ligated for 45 minutes at 22°C. So far I've had no positive colonies.
Does anybody have any idea about how to make this work?