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weird bands after minipreps - molecular cloning (Feb/08/2011 )

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?


What percentage gel are you running? It could be that the 11 kbp band is stuck in the wells.


My gels are 0.8% agarose. And I see the higher band when I run my ligation reaction on the gel.