info on endotoxins

[Jeswin]

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

[Sebastian S. Cocioba]

Sydlabs has endo-free silica tubes complete with kit for 10 preps at 160 bucks.

http://www.sydlabs.com/ProductDetails/4-19-152-1356/endofree-plasmid-maxi-kits-spin-column.html

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

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[Max Berry]

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.

[Jeswin]

On Tue, Feb 5, 2013 at 1:58 AM, Max Berry <maxb...@gmail.com> wrote:
> 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.
>

So after I eluted from our maxiprep column (in Buffer QF, before
isopropanol precipitation), I added 200uL of diluted triton. I let it
incubate on ice 10 minutes, then at 37C for 20 minutes. I centrifuged
it down since I thought I saw a phase separation. Turns out it was not
a phase change I was looking at but due to the Falcon tube bending
light or something. Anyway, turns out there was no phase change.

I think it may have something to do with the Buffer QF* components. I
ran a test beforehand using 50uL DNA in 450uL TE and I was able to see
the phase change. Is higher temperature needed to get the phase
change? I read something about the cloud point changing if other
chemicals are in the solution.

Anyway, we're running out of time so we're just going to precipitate
the DNA with isopropanol and elute in water. Afterwards, we'll try to
do the endotoxin removal step.


*Buffer QF (elution buffer)
1.25M NaCl
50 mM Tris-HCl pH 8.5
15% isopropanol

[Nathan McCorkle]

On Wed, Feb 6, 2013 at 1:00 PM, Jeswin <phill...@gmail.com> wrote:
> I centrifuged
> it down since I thought I saw a phase separation. Turns out it was not
> a phase change I was looking at but due to the Falcon tube bending
> light or something. Anyway, turns out there was no phase change.

I don't think he mentioned that you'd see a phase separation... I
think he probably just meant that the endotoxin is lighter than DNA,
therefore it will be in the top part of the solution.

I could be wrong, I guess we'll have to hear back from Max

--
-Nathan

[Max Berry]

Yeah the 15% isopropanol is definitely gonna keep the triton soluble so it wouldn't separate - I forgot Qiagen uses isopropanol in those buffers. If you do resuspend in water, the phase separation protocol should work in that, or you can add a little triton into the load before you put in back on the column, which might help on the next run. Actually, it might well have separated at 37C even with the isopropanol, but if your centrifuge isn't heated then it might have cooled down and resolubilized again.

[Jeswin]

On Wed, Feb 6, 2013 at 6:18 PM, Max Berry <maxb...@gmail.com> wrote:
> Yeah the 15% isopropanol is definitely gonna keep the triton soluble so it
> wouldn't separate - I forgot Qiagen uses isopropanol in those buffers. If
> you do resuspend in water, the phase separation protocol should work in
> that, or you can add a little triton into the load before you put in back on
> the column, which might help on the next run. Actually, it might well have
> separated at 37C even with the isopropanol, but if your centrifuge isn't
> heated then it might have cooled down and resolubilized again.
>

I see. Thanks for the input. I don't understand what you mean about
the heated centrifuge? I centrifuged at room temperature (25-27C) so
you're saying that it resolubilized then? I didn't really notice any
phase after taking it out from the 37C but maybe I wasn't paying
enough attention.

[Max Berry]

I just meant that you may have had to keep the sample at 37C to keep the phases separate during centrifugation, but that's assuming they even existed in the first place. I'm only guessing, since I've never considered running the protocol with alcohol in the solution - it probably did just keep the Triton soluble the whole time.

Oh and regarding Nathan's post, the Triton+endotoxin will definitely be in the bottom layer, not the top.

[Eugen Leitl]

On Wed, Feb 06, 2013 at 04:00:33PM -0500, Jeswin wrote:

> Anyway, we're running out of time so we're just going to precipitate
> the DNA with isopropanol and elute in water. Afterwards, we'll try to
> do the endotoxin removal step.

Which parts of your experiment are sensitive to endotoxins?

[Jeswin]

On Mon, Feb 4, 2013 at 3:44 PM, Sebastian S. Cocioba <scoc...@gmail.com> wrote:
> Sydlabs has endo-free silica tubes complete with kit for 10 preps at 160
> bucks.
>
> http://www.sydlabs.com/ProductDetails/4-19-152-1356/endofree-plasmid-maxi-kits-spin-column.html
>

Have you used Syd Labs products before? How are they? Qiagen has the
market so I never heard of these other companies, except Promega.
Qiagen sells their for $30/prep vs Sydlabs $16/prep.

On Thu, Feb 7, 2013 at 5:26 AM, Eugen Leitl <eu...@leitl.org> wrote:
>
> Which parts of your experiment are sensitive to endotoxins?
>

No idea. Its part of a contact we got hired to do requesting
low-endotoxin, high supercoiled plasmids. I think I'll take a look at
the Syd Labs kit. It will save us the time next time we have to do
this.

[Dakota Hamill]

Well Sydlabs is cheap, but they never have anything in stock.  I tried to get 5 different things and they said they were all out...never told me when they'd have them back.  Maybe they're out of stock because their prices are so good.  But I doubt it.

[Jeswin]

I see that they have 1-10 employees. Maybe they only produce in small
batches and don't keep much in stock.

[Jeswin]

On Wed, Feb 6, 2013 at 7:02 PM, Max Berry <maxb...@gmail.com> wrote:
> I just meant that you may have had to keep the sample at 37C to keep the
> phases separate during centrifugation, but that's assuming they even existed
> in the first place. I'm only guessing, since I've never considered running

Max, I got some weird result in my nanodrop. In a test of DNA with
sodium acetate added, the concentrations after triton extraction are
greater than initial concentration readings. I think something is
interfering with the reading, maybe the salt. Or maybe the salt
changes something so that triton is not fully removed? Any ideas?

Thanks

[Max Berry]

I'm not totally sure how you mean you prepared the samples, but Triton will absorb strongly in the UV. How did you go about removing the Triton?