Transformation efficiency? - (Apr/30/2007 )
I have a problem! I am using the pet-15b system to clone my gene into chemically competant E.coli Bl21 strain. I amplified my gene with a single BamH-1 site on both ends using specific primers. I cut the vector with the same enzyme. Then I dephosphorylated and used the purified vector to ligate with my gene. This i transformed into E.coli. and I plated the cells and inoculated the remaining culture in broth with the selective antibiotic. The plates were empty but I got growth in the broth. I then isolated plasmid from the broth culture and I cut it with BamH-1 . I used the plasmid to also PCR the gene using specific primers. When I ran a gel of these two. I noticed that the restriction digest showed just one band instead of two and it was of the size of the vector, whereas the PCR showed the band of my gene. I dunno if my gene has been ligated into my vector. How do I verify? What is going on here? Can any one come up with an explanation for this? I am so confused. Please help.
Do your primers amplify the junction area between the vector and insert. Ie one primer binds to the vector and the other primer binds to the insert. This PCR signal is more trust worthy as the vector-insert junction is unique.
If the primers bind only to the insert, any free insert DNA floating about in the recovery media (which it will) could theoretically survive the plasmid extraction protocol and give you a positive signal on the PCR. PCR is very sensitive.
Alternatively the BamHI digest might have gone funny. In which case you could redigest your DNA.
However do note, it takes only 1 cell transformed by a single religated vector molecule (which defeated the odds and survive dephosphorylation) to turn your both cloudy. In my opinon it is very likely there is nothing in the culture aside for vector.
What would a BamHI digest on vector molecule look like? A single band?