Reasons for failure of transformation - (May/24/2011 )

I'm sorry I didn't give more information:

Vector: pET28a(+)

Insert: PCR amplicon (I included the restriction sites plus 5 more nucleotides on either side of the primers)

Restriction enzymes: NcoI and XhoI

Cloning host: E.coli DH5alpha

Antibiotic selection: 100ug/mL Kanamycin

I double digest the vector and insert overnight at 37oC, run an agarose gel and elute the digests from it. I then carry out ligations with the gel eluates overnight at 16oC. When I transformed the competent cells (They're good preps because I always run an uncut vector transformation as a control), I got about 10 clones which I then transferred from plates containing Kanamycin to broth containing the same amount of Kanamycin. There was good growth overnight at 37oC and so I proceeded to isolate the plasmids from them. That's where the problem began -  I get a good plasmid prep but when I digest the plasmids, I can see that there is digestion but the bands are way lower than where they should be (almost at the size of the insert). I can't be transforming insert dimers because then the cells would simply not grow in the presence of Kanamycin.

The only difference I see when I isolate the plasmids from the "recombinant" and non-recombinants is that I always see the nicked plasmid band in the non-recombinant, but never in the "recombinant". Always.

It's clear therefore that I am missing something but I can't figure out what. I've checked that both my restriction enzymes and my ligase (and buffers) are working with lambda DNA.

I suspect that the plasmids you are recovering from the Kan plates are either the original uncut plasmid, or (more likely) ligation of a PCR primer-dimer into the cloning site. In both cases, you will see small fragments when the plasmid is cut, and no insert. Is the gel for your pcr product showing a clean band with little or no short fragments showing? If not, then I suggest you concentrate on improving your pcr reaction (change annealing temperature, redesign primers). You might find the primer analysis tools at IDTDNA.com useful. Particularly look at the heterodimer results.

Are you doing a control where you ligate and transform without the insert present? This would be useful in determining what is happening.

You can't tell anything about the quality of your competent cells by transforming uncut plasmid, unless it is diluted to 10-100 pg/ul level, and transformed with 1 ul.