Posted 08 February 2011 - 10:56 AM
I am trying to subclone a 1.5kb NotI-NdeI-cut fragment into an 11kb vector. I gel purified both vector and insert, and ligated using T4DNA ligase, overnight at 16 degrees. I used single use competent cells from invitrogen for transformation. The control plate (cut vector only ligation) had very few colonies and the plates from vector-insert ligation had 100s of colonies. But when I performed minipreps, cut plasmids with NdeI and NotI, I see two bands on the gel, one of them is 3kb long and the other is 1.5kb long. I don't see a higher 11kb band at all. What is happening here? Where is my vector?