I want to know if anyone has ran into this problem before: I'm making a deletion construct and I have positive results in colony screening. When I miniprep the plasmid after overnight culture, the plasmid is no longer there. What am I doing wrong?
Your question is too vague. What do you mean by having positive results in colony screening? Just that you get colonies when plating the transformed ligation reaction or that those colonies you do obtain are positive by e.g. colony PCR? Also, do you also have problems with the original plasmid, or only with the putative mutants?
I'm having problems with the putative mutants. After completing a ligation and electroporating the new vector + insert into competent cells, plates are made with the appropriate antibiotic. Colonies are picked and colony PCR is performed with the vector primers as well as the insert problems to ensure the colony has the plasmid. These results are positive, then the colonies are used for an overnight culture. A miniprep is performed and when a gel is run (before sequencing) neither the plasmid or insert band appear on the gel with vector primers, or insert primers.
Oh, I see. So I take it the minipreps from the deletion fail to yield DNA (as assessed by electrophoresis of the minipreps themselves, rather than PCRs run from them) but minipreps from a transformation of the unmodified plasmid performed in parallel work perfectly fine?
Hi Gregory,
1. Has you added the same antibiotic into overnight bacterial culture to maintain the presence of plasmids in the new divided cells?
2. Did you follow the exact steps of the protocol for miniprepping? You can miniprep a control side by side with your sample. This will check if your miniprep kit is ok.
Hi!
No I still do not have a successful plasmid...although I am hopeful. I started over with new reagents and have had brighter and more isolated bands. Other folks have used the miniprep kit and got fine results, and I did as well with another construct. Antibiotics were added as well.
I noticed that you are doing a 'deletion' construct. Do you think that this 'deletion' might cause impact on the disappearing of your plasmid in liquid medium culture (but not on solid medium culture)?
I'm not sure...it is an in frame deletion, so it should only be deleting the tryptophanase gene.
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