One possibility is that the checking of the insertion by pcr is giving the wrong answer. If the pcr primers are both inside the insert then wetting of the plate with ligation mix will give a positive pcr from the small amount of ligation mix spread over the plate. Later testing after colony growth will be negative because the ligation failed. The best primers for insert are one primer in the insert and the other primer in the vector

If you are getting some of the plasmid re-circularized without the insert, and the insert is somewhat toxic on the host cells, the insert-free plasmid may replace the one with insert after several growth cycles. Even if the encoded protein is not expressed at all, the insert may increase the burden of plasmid maintenance just because of its size. Or it may contain motifs which cause problems upon plasmid replication. This way, the insert-free plasmid would be more beneficial for the host, and will eventually take over the population.

Another possible scenario is the loss of insert from some of the plasmids by recombination (check if you have sequence repeates near the ends of the insert). Afterwards, the insert-free plasmid spreads through the host population the same way as described above.

You may get some clues on it by running two PCRs on you plasmid preps. The first is the one which you already use, with forward primer on the plasmid and reverse primer on the insert. The second one is with both forward and reverse primers on the plasmid on the flanks of the insertion site. The first PCR would tell you if you have the insert in right orientation. The second PCR would show how much do you have the plasmid with insert versus the plasmid without insert (qualitatively, of course).

Hi Oh,

do you have a positive control for your PCR to make sure that the PCR is working? From your description it seems that you did your PCR directly on the bacteria you have grown over night.

If this is the case I would recommend to do a plasmid preparation and try the PCR directly on the plasmid. If you have done this already I would check the the template concentration, because using to much of the template occurs quite often and could interfere with your PCR - 10 ng for a plasmid should be enough (and include a positive control).

As Victor G Stepanov recommended it might be good to do two differnt PCRs with insert and plasmid primers.

However, what I also would recommend is to do a digestion with restriction enzymes flanking your insert to see if you can cut out the insert. You might also search for an enzyme which cut in the insert and in the vector (one cutting site in the vector and one in the insert - if there is one), because this can give you already a hint if you have the right insert and also about the orientation. If everythings fail I would sequence the plasmid again.

Never rely ONLY on PCR results; do also the "old school" minipreps and some restriction digest (or at least on your maxi DNA prep) and don't rely on glycerol stock; DNA is a much better for conservation (you can sequence it and only nanograms are required to do a new clone) and it will work even if your frezer breaks down.

I second Dr. Poncet's point. When I was a postdoc, about 1 in 10 of my plasmid-transformed glycerol stocks would lose the plasmid, regardless of what confirmation approach I used. This also happened to others in the lab occasionally. I completely stopped making glycerol stocks; now I keep plasmids at -20C as minipreps. Retransformation only takes one additional day (transform, grow colonies overnight, grow liquid culture overnight). Doing this also saves us a lot of space in the -80C freezer.

Hi — this happens more often than you’d think. Two common causes are (1) selection for plasmids that lost the insert (especially if the insert is toxic or leaky under Ptac/LacI), and (2) selection/antibiotic problems or PCR inhibitors from your glycerol culture.

Quick diagnostic steps: plate the glycerol stock on fresh kanamycin plates and pick multiple single colonies; do colony PCR with (a) insert-internal primers and (b) vector-flanking primers to detect deletions; perform minipreps + restriction digest on several colonies; and PCR for the kanR gene to confirm the backbone. Also ensure your kanamycin stock is fresh and at correct concentration, and grow with glucose (to repress Ptac) if leaky expression might be toxic.

If you still get no insert in all colonies, it’s likely the insert is being lost (deletion) during outgrowth — use repressed growth conditions, a lower-copy vector, a lacIq host, or remake clones and freeze multiple fresh glycerol aliquots. If you want, share a gel/photo of your colony PCR/miniprep digest and I’ll help interpret.

I had this issue once, my bactera was conjugated and had this plamid that expressed a resistance cassete and had my gene of interest. The bacteria had a specific phenotipe. Everything was good untill I made glycerols. Once I tryed to work with the bacteria again, it lost its phenotipe. The PCR would show the gene from the plasmid, but for watever reason it did not express the gene. So I had to do the conjugation all over again. So what I learned from this specific bacteria was that it always lost the expression of the gene of interest once it went to glycerol. So everytime I needed this particular bacteria with this gene, I would have to make the whole thing over again (insert the fresh plasmid with the gene of interest into the bacteria by conjugation).