genomic DNA contamination during plasmid preparation - (Dec/17/2007 )
shy to ask this question as I have worked on this for years. although I know the principle well, and have done this experiments many times, I do not know why recently, I have failed twice.
I have prepared a vector DNA by using QIAGEN midi kit, good quality, and then inserted another cDNA, after I finished the cloning and sequence confirmation, I transfored the succeeded DNA (recombinant DNA) to DH5a, and want to prepare midi amount of DNA by using the same kit. then for twice, from 100 ml of bacterial culture, I got no plasmid DNA at corresponding size, while only genomic DNA at the position corresponding to marker of 10,000 bp.
I do not know why, when I measured the OD, they are good, between 1.8 and 2.0. concentration is around 0.5 ug/ul.
I have done this experiments for so many years, I do not know what is the problem.
whether the inserted cDNA could be a reason? no, because when I screened the positive recombinant DNA, I purified many plasmid from the transformed product, and got no problem. then why now>?
thank you if you can give me an explanation.
P.S., only 1 band appear in my gel, around 10000 bp, for sure it is not my target plasmid DNA>
I think there is only chromosome DNA but no plasmid DNA in my samples, why? and very few DNA pellet after precipitation with isopropanol.
I had a similar problem recently. The sequence I am working with is highly repetitive so the bacteria (DH5alpha) do not like it very much. I just left my culture overnight in the fridge and next day I did not get any plasmid. Growing a second midi culture from another positive miniculture did not help either.
So I did the transformation again, growed cultures right after (~12h) and did the prep. Then it was fine.
Was the prep from fresh transformation/culture when you tried it the second time? Or the other possibility is of course that there is something wrong with the kit. But then others in your lab would also have some problems.