2 replies to this topic

[mouser]

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?

[bob1]

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

[mouser]

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