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Transformation and ligation problems - (Dec/18/2005 )

I have a problem with E.Coli transformation
I transformed cells with plasmid that should contain 2,2 kb insert
Actually there are almost twice as much colonies in my positive control (digested plasmid) than in "normally" performed ligated fragments
What does it mean?
What should I check?


I couldnt get ur question clearly.

Anyway I'll try to clarify.After digesting vector go for back ligation and check background colonies.If background colonies are zero,the digestion is good, then the colonies u get after ligation of intrested insert will have the same.

U can screen for ur insert by simply digesting withe available enzymes in Multiple cloning site(MCS)
of the vector.



Your control plate should contain only the vector and not insert but with other ligation ingredients. So ideally no of colonies on control should be less. By saying you have digested dna in control, do you mean you have digested the ligated product with such an enzyme which will cleave only if digestion has not occured!(say x site will close after successful ligation)!. I hope I didnot confuse you more huh.gif


Had the same problem with my transformations. You should be very sure that all the restriction enzymes you used work. In my case one of the two enzmymes (though freshly purchased) didn't cut properly and so after ligation I got lots of colonies of the religated 'negative-control' (the cut vector) but less on the normally ligated ones.