I wonder what plasmid purification kit are you using. I need some, that is endotoxin free, as I am interested in studying cellular stress, suitable for scale up ,as I need 100ug for one special experiment now and that gives high yeild of DNA. Satisfactory concentration for me is at least 1000 ng/ul for pEGFP plasmid. I tryied Renogen Biolab kit, it is very quick and convenient when doing mini preparation but it is hard to scale it up. When I tried to use it for gigapreparation I didn´t succeed at all as the DNA binding resin is very hard to resuspend in that amout (1ml) and I think I lost most of the DNA by extensive vortexing of the resin to be able to wash it. Many considers Quiagene to be the best, but endofree Quiagene is very expensive. I could use normal Quiagene, but I am afraid, there might be some endotoxin left :-( Do you have any ideas or interesting experiences? Thank a lot in advance, Klára
We always use PureYield™ Plasmid Midiprep System.The Endotoxin removal wash is supposed to remove protein, RNA and endotoxin contaminants to improve the efficiency of downstream applications such as eukaryotic transfection, in vitro transcription and coupled in vitro transcription/translation. Essentially, the kit removes lipids which can induce immune response in mammalian cells which may negatively affect our experiments.
We always use PureYield™ Plasmid Midiprep System.The Endotoxin removal wash is supposed to remove protein, RNA and endotoxin contaminants to improve the efficiency of downstream applications such as eukaryotic transfection, in vitro transcription and coupled in vitro transcription/translation. Essentially, the kit removes lipids which can induce immune response in mammalian cells which may negatively affect our experiments.
PureLink Expi Endotoxin-Free Mega/Giga Plasmid Purification Kit - from Invitrogen
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