I'm guessing this is for a purified sample post-maxiprep, in which case you can add a small amount of Triton X-114 (like, 1%) and mix on ice, then centrifuge at room temp and pull off the top layer, which contains your now-depyrogenated plasmid (bottom layer is Triton + endotoxin). However there'll still be quite a bit of Triton in the upper layer, so traditionally you'd repurify - assuming that whatever's sensitive to endotoxin won't particularly like Triton much either (it won't). Your re-purification should then be clean unless you introduce new endotoxin.
Qiagen's super-secret buffer ER for endotoxin removal is commonly believed to contain a small amount of Triton X-114 in solution, such that when it's mixed with your load and run through the column, it will carry the endotoxin out with it, then you wash and elute as normal. This work just as well with Triton X-100, which is somewhat easier to find. In fact, don't bother with the first method I mentioned since if you're gonna end up doing a re-purification anyway, just mix in a little Triton with the plasmid, stick on ice for a while, and re-run on the column as normal (make sure to dilute the load if it's in high salt buffer).
You can also try doing a 20% ethanol wash between loading and washing, which can be effective at removing endotoxin as well, as long as the column is anion exchange and elutes with high-salt buffer.
Kits for endotoxin detection have quite a high baseline for cost, since I don't think anyone's made one that doesn't require delicious horseshoe crab blood. The active ingredients tend to last in the fridge for a couple years if they're lyophilized though.
On Monday, February 4, 2013 10:14:47 AM UTC-8, phillyj wrote:
Hey guys,
What can you tell me about endotoxin removal from plasmid maxiprep?
Anyone know of the protocol for using Triton X-114 to remove
endotoxins? It's some sort of aqueous phase separation. I'm not too
sure.
Is there a cheap way to detect endotoxins, e.g., before and after
purification? The gel-clot LAL assay is probably around $200 for 20-30
assays but I just need to test 7 samples.
Thanks
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