Hello,
I am working with endotoxin sensitive cells now and electroporation is quite toxic to them compared to an endotoxin free control. So I think I am going to clean up my existing plasmids with triton x-114 which seems easy enough.
But I would also like to do my future preps endotoxin free/reduced but have a big kit left that's not "endo free" and I don't want to buy a new one.
Has someone experience with using Triton X-114 during (e.g.) midi prep and could elaborate a bit? I read about two different methods:
1. Add Triton after neutralization+centrifugation and clean up with cool/heat cycles
2. Add Triton to the wash buffer, before elution (which seems too easy to be true to me)
I am using a standard Midi Kit with standard buffers:
Cell Resuspending Buffer (E1) 50 mM Tris-HCl, pH 8.0 10 mM EDTA RNase A 20 mg/mL Lysis Buffer (E2) 0.2 M NaOH 1% (w/v) SDS
Precipitation Buffer (E3) 3.1 M Potassium acetate, pH 5.5
Equilibration Buffer (E4) 0.1 M Sodium acetate, pH 5.0 0.6 M NaCl 0.15% (v/v) Triton X-100
Wash Buffer (E5) 0.1 M Sodium acetate, pH 5.0 0.8 M NaCl
Elution Buffer (E6) 0.1 M Tris-HCl, pH 8.5 1.25 M NaCl
I'll be happy to receive your input!
You can use Triton X-114 at the last step, i.e., add Triton X-114 to eluted DNA solution. Then heat it up, and Triton X-114 will precipitate at high temperature.
I would add the Triton X-114 at the wash step, then wash extensively with Triton-free and exonuclease-free wash buffer to get rid of any Triton, followed by endonuclease-free elution buffer. Normally, the buffers supplied with the kit should be endotoxin-free, but in case they are not, you could try to purify them using a polymixin column. Most of the endotoxin is probably coming from the lysed E. coli host in which you grow your plasmid, and not from the buffers.
The problem with Chengkang's suggestion is that even though Triton X-114 will separate and form a phase at high temperature, there may be some of it left in the aqueous phase, which may be harmful to your cells
Hello Pierre,
to clarify this, should I:
- after neutralization + centrifugation cool down the crude DNA extract on Ice
- add 1% Triton and mix
- heat to 55°C and precipitate by centrifugation at RT
- Load the supernatant to the anion exchange column
- Wash with 10ml wash buffer (more than 2x ?) ?
Would that work?
Thanks
there are alternative protocols, detergent-free, and allowing to obtain low endo or endofree plasmid DNA in just 30 min. eg:
https://www.thermofisher.com/order/catalog/product/A36228
Alexander. I routinely use these kits and they produce "low endotoxin plasmid (
There isn't such thing as endo-free plasmid preps but treatments mainly with detergents at the early stage of the plasmid preparation process can significantly decrease the amount of endotoxin in your final product. If you need to prep your plasmids with a low endotoxin level, forget about the DIY kit option! Send us your plasmids as we can prep them for only a fraction of the price offer by other companies. You actually save money if we do it for you! By using our own proprietary protocols, we can not only offer the best price to our customers but also obtain better results than you would with a kit in terms of DNA quantity and quality. See for yourself at https://www.biozilla.bio/plasmid-dna-purification
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