Hello, I just wanted to know if anyone has ever experienced anything like this, where transforming DH5-alpha cells with DNA results in the DNA itself being modified?
I have checked the sequence of my transforming DNA (plasmid is pMSCV, AMPr) and confirmed the presence of my mutations (no signs of a mixed DNA population, the electropherogram is clean).
However after using this DNA (first time by moving the DNA into a clean, new Eppendorf tube, second time by adding competent cells directly to the original tube with the DNA) to transform DH5-alpha cells, I continually somehow get the wild-type version of my gene of interest from kit minipreps. There is no sign of a mixed DNA population, the minipreps yield wild-type DNA with no mutations present.
The competent DH5-alpha cells alone do not contain ampicillin resistance and no colonies formed on my negative control plate. I'm 100% certain I'm using the correct DNA. Not sure how this is possible, but I suspect it's happened previously before with this specific mutation in this specific plasmid (pMSCV), by someone else. There is no -80 degree bacterial glycerol of this mutant (the only one made previously turned out to be wild-type too).
If anyone has suggestions on what might be happening here, I'd appreciate hearing them.
Have you tried to do this with mutant clones obtained in the secondary transformation. Possibly you can find a stable clone among them.
Hello Sanna
While cloning for site directed mutations, we prefer using SURE cells (http://www.genomics.agilent.com/article.jsp?pageId=664), since in Dh5 alpha we tend to observe something similar to your case.......I cannot pin point the reason but it maybe some correction mechanism inherent to the organism.
Good Luck
Solved this issue a while ago and thought I'd post the answer. The problem most probably was that the tube had two kinds of DNA (WT and mutant) mixed together to begin with. It wasn't obvious based on sequencing of the DNA initially, but after transforming the same DNA, the new colonies all contained either only WT or a mixture of WT and mutant, according to the electropherograms.
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