When I plate Mach 1 cells transformed with plasmid containing no insert on LB plates+ Kanamycin I see very little number to no colonies growing. But when an insert is present in the plasmid, the plate will be full colonies. It may be a trivial question, but WHY does this happen? Cheers, Paolo
Please check the concentration of your plasmid (vector) control. It is possible that the amount of vector you are using for transformation is lesser than the Plasmid+insert DNA.
If there are no issues in the concentration, the other possibility is that, the insert might give some growth benefit to the E.coli cells growing in selection media. May I know your cloning fragment?
Question: What genes are on your vector backbone, besides the resistance marker? Is it a zero-background vector with a "toxic" gene that is disrupted by insertion in the MCS? Sounds suspiciously like something like that...
Hello Guys the question here is a more theoretical one. I think I got the answer anyway. It has to due with the fact that when I digest the plasmid at two different restriction sites and then I perform ligation with no insert, then the plasmid will not be able to ligate back and it will therefore not be circular DNA plasmid but just a stretch of DNA. I think that this will affect the transcription of the antibiotic resistance gene and therefore the transformation efficiency.
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