I think you should review your steps, did you include antimicrobial assay stage? I don't really know what you are doing but maybe you should allow more time in the ligation step. Check the resistance gene the plasmid is using and the appropriate medium for cell cultivation. Did you design your own plasmid or commercially bought? Who knows there might be less sequence stringency. What I'm saying is the problem can arise from any of your steps so try and review and if possible try a different ligation enzyme.

Hope it helps!

Ken.

Thanks Ken. Well, the plasmid that i used as back bone has been amplified from a modified plasmid. My insert is about 72 bp. I think my insert did not ligate in or either that my amplified plasmid might be the problem. I did optimized the ligation time to overnight incubation at 4 Celsius. I practiced culturing my cells in 2yt medium.

But if your insert did not ligate then how come you got some colonies in the first place to work with? Don't you have any step to assay the colonies of interests? For example, I use plasmids with selectable markers that help me to distinguish between colonies with the recombinant plasmid; cells that don't pick plasmids die in the antibiotic incorporated media, those that pick non-recombinant plasmid survive but of no importance to me because they don't have my gene but I'm able to distinguish them because the produce a color on the growth media, the non-color producing colonies are my cells of interest because they have my gene. I don't really know the method you using, maybe a modified one after all but at least to eliminate the ligation step from your sources of error, i think you should have some sort of assay to confirm that the gene of interest was transformed in the cells in the first place.

Have you ever used this amplified plasmid in any successful work? This will help you to eliminate the amplification procedure as a source of error.

In what solution was the 4 celsius overnight incubation done? and if you can get an electroporator, you can backup that transformation step to see where the problem lies. But check the ligation step first since i think it's the primary source of focus. I just want you to have a successful experiment because it hurts when you don't get what you expect in the lab.

Ken!