positive colony PCR, negative restriction digest, positive PC from minipreps - Cloning nightmare (Mar/05/2011 )

I've been attempting directional cloning, putting a 1.6 kb gene into an 8.5 kb vector with NotI and AgeI sticky ends, and transfecting DH5a cells. I get no colonies on empty vector ligation plates, so hopefully my double digestion of vector worked and I see no re-ligation of empty vector. I got a few colonies with my gene insert present in the ligation, so did colony PCR with primers to detect the insert. 6 of the 8 colonies were positive so I grew them up and miniprepped out the plasmids, alongside one of my negative colonies as a control. I did two restriction digests, both of which showed ever plasmid to be 8.5 kb, and not containing my insert. To confirm this I repeated my diagnostic PCR, this time on the purified plasmids, and I still got 6 positives and one negative, and my negative control of empty vector was indeed negative. What on earth is going on? How can I have insert present if the digest is not producing bands expected from insert-positive vectors, and is showing linearised vector at the size they would be if they were empty?

Is your colony PCR amplifying just the insert, or from primers surrounding the cloning site? If it is just the insert, then carryover from the ligation onto the plate can result in positive amplification. Is the insert showing the correct length in the PCR reaction? You might be inserting a short fragment which amplifies, but is sufficiently small to not show up on a gel.

Are you sure you are not missing the 1.6 Kb band on your gel? It will be a lot weaker than the 8.5 Kb band, and if you use a gel with EtBr mixed and fail to post-stain, the band might be further weakened by migration of EtBr toward the negative electrode.

phage434 on Sat Mar 5 15:35:29 2011 said: