Iíve been trying to clone a few geness into pET19b using RE sites NdeI and BamHI. I first digest overnight with NdeI, heat inactivate at 65 degrees for 20min, then digest with BamHI for 4hr and CIP for 1hr before gel purifying on a 0.8% agarose gel. The gel always looks good when compared to digested and undigested control vectors (I see 2 bands at expected sizes for the linearized DNA).
For ligations Iíve been utilizing Quick Ligase with a 3:1 ratio of insert:vector. My controls have been the same as the ligations, minus insert, and only yield 1-2 colonies at the most, while my ligations have many (20-30) colonies per 50uL of C43 DE3 cells plated. However, when I pick colonies for minipreps, most of the colonies run lower than, or even with, the undigested pET vector. Iíve even digested the miniprepped samples (thinking maybe nicked v. supercoiled DNA maybe responsible for the difference) and found no insert.
Has anyone worked with pET vectors and encountered similar issues? Iíd appreciate any advice, thanks!!!