Problem with genomic DNA contamination in Minipreps - Qiagen QuickLyse Miniprep Kit (Oct/24/2008 )
Hi, I have been doing this miniprep (the Qiagen QuickLyse miniprep kit) for almost 2 weeks now, and have consistently gotten a genomic DNA (could be RNA too) smear upon restriction analysis. I use TOP10 cells, and have tried the miniprep on both my own plates as well as a colleague's. I have vortexed a lot and also not vortexed at all, as the suspicion was that too much vortexing was shearing the chromosomal/genomic DNA and contaminating my prep. The buffers are all correct, and have been used by other members of the lab with success. Does anyone have any suggestions? This is really hindering my experiment. I have previously done many successful minipreps; only in the past two weeks has it given me any problems.
Any help is appreciated! Thanks.
Any help is appreciated! Thanks.
If you just run the plasmid uncut what did u get? smear as well? coz sometimes restriction enzyme could be the culprit.
and i don't think i vortex my sample when doing miniprep.. just invert a few time would do.
You should never vortex a miniprep. The idea is to gently lyse the cells so the small, numerous plasmids "leak" out, while the bulky chromosomal DNA remains trapped in the cell debris. Work gently and quickly throughout the prep, especially the lysis step -- as Hanming86 mentions, a few inversions are sufficient to mix during the lysis step, then straight to the neutralization step -- no vortexing, no prolonged incubation, no pipetting up and down.