Phenol-ether plasmid extraction?
Meredith L. Patterson
minipreps. Chloroform and ether have rather similar solvent
properties, but whereas I can easily get phenol and ether (they're
available OTC in pharmacies in Belgium), chloroform is restricted. I'm
looking into what I'd need to do to be able to purchase chloroform
legally here, but naturally if ether works (even if yield is lower),
that will be simpler.
Google isn't yielding up much on the subject of phenol-ether plasmid
extraction, as ether is sometimes used in other steps of a miniprep.
Anyone have any experience/advice to lend? What solvent property of
chloroform makes it useful in plasmid extraction? I gather that the
P/C extraction step is to remove protein fragments, as some protocols
apparently just use phenol there; what role does chloroform play, when
it's used?
Cheers,
--mlp
Cathal Garvey
I'll ask around in my lab tomorrow about Ether extraction. I imagine it can't be that different to Chloroform. Sure you can do DNA extraction using dishwasher detergent and cold rubbing alcohol, I'm sure something like Ether would do the job just fine.
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Meredith L. Patterson
> I didn't know you were Belgian!
Well, not yet. It'll be a few years before I can apply for
citizenship, but I live in Leuven.
> I'll ask around in my lab tomorrow about Ether extraction. I imagine it
> can't be that different to Chloroform. Sure you can do DNA extraction using
> dishwasher detergent and cold rubbing alcohol, I'm sure something like Ether
> would do the job just fine.
Thanks! In particular I'm looking for alternatives to
phenol/chloroform extraction for plasmid miniprep on Gram-positive
bacteria (which typically involves pretreatment with lysozyme, and
presumably a lot of protein crap lying around in need of removal). OWW
suggests that lysozyme followed by a normal Qiagen miniprep will work,
though I don't see any reports of experience with this approach other
than a reference to Mathiesen et al. 2004.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC182348/ is a miniprep
protocol from 1993 which uses P/C -- when my pharmacist gets some more
phenol in I'd like to try this on lactobacilli using P/E (and perhaps
straight P and lysozyme/column for comparison) rather than P/C unless
it's simply unlikely to work at all.
Cheers,
--mlp
Brent Neal
Meredith -
My guess is that chloroform is useful mixed with phenol because it is
a fairly polar solvent that is miscible with damned near everything.
Phenol is relatively nonpolar, so as a cosolvent mixture I can imagine
them solvating amphiphilic things like proteins very well. You should
look for other polar solvents to replace it.
Or, if you are feeling pretty ambitious, look up the haloform
reaction. If you have a distillation column, you could make chloroform
yourself from acetone and bleach. :) I don't think I'd recommend this
route, because I expect you will need fairly dry acetone to make it go
and some of the other potential products of that reaction are powerful
lachrymators. (Chloroacetone, e.g. is WWI era tear gas.)
Not being an expert on EU law, I know that if you order your solvents
through a corporate entity in the US, you can avoid the legal issues.
Perhaps such a route is open in the EU as well?
B
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http://brentn.freeshell.org
<bre...@gmail.com>
Meredith L. Patterson
> My guess is that chloroform is useful mixed with phenol because it is a
> fairly polar solvent that is miscible with damned near everything.
Wait, what? Chloroform is highly nonpolar, with a dielectric constant
of 4.8 (compared to water's 80). Diethyl ether has a dielectric
constant of 4.3 (though
http://en.wikipedia.org/wiki/Solvent#Properties_table_of_common_solvents
lists diethyl ether as slightly more polar than chloroform -- not sure
what's up with that).
But the point of the liquid-liquid extraction technique is to use one
polar solvent and one nonpolar solvent, so I'm assuming that phenol is
polar and ether very close to chloroform in terms of (non)polarity.
> Or, if you are feeling pretty ambitious, look up the haloform reaction. If
> you have a distillation column, you could make chloroform yourself from
> acetone and bleach. :)
Thanks, I think I'll pass on that one until I get my hood built ;)
> Not being an expert on EU law, I know that if you order your solvents
> through a corporate entity in the US, you can avoid the legal issues.
> Perhaps such a route is open in the EU as well?
Probably, though if I can develop a protocol that works with wholly
OTC materials I'll be happier.
Cheers,
--mlp
Brent Neal
<clon...@gmail.com> wrote:
> On Mon, Jan 25, 2010 at 6:29 PM, Brent Neal <bre...@gmail.com> wrote:
>> My guess is that chloroform is useful mixed with phenol because it is a
>> fairly polar solvent that is miscible with damned near everything.
You're exactly right - I thought one thing and typed another. :P Sorry.
>
> But the point of the liquid-liquid extraction technique is to use one
> polar solvent and one nonpolar solvent, so I'm assuming that phenol is
> polar and ether very close to chloroform in terms of (non)polarity.
An easy way to eyeball it is to look at charge density. Chloroform is
trichloromethane. Chlorine, like all halogens, really wants your
electrons. but with three of them, the sum of the dipole vectors is
pretty short. Only way for it to be shorter is to have
tetrachloromethane and the vector be zero. On the other hand, the
oxygen in the alcohol has two nice lone pairs, and with most alcohols
and ethers, this leads to a dipole moment and a reasonably polar
solvent. Phenol is probably moreso, but the delocalized electrons in
the ring might have a confounding effect. I'd have to think about that
a little more.
Mind you, looking at it this way doesn't tell you everything, but it
is correlated and it can give you some intuition about how polar a
solvent is and is empirically good. Using, e.g. the halomethanes as an
example, you find that the Hansen dP for dichloromethane is about 6,
chloroform is about 3 and tetrachloromethane is generally reported as
0, as you would expect.
>
>> Or, if you are feeling pretty ambitious, look up the haloform reaction. If
>> you have a distillation column, you could make chloroform yourself from
>> acetone and bleach. :)
>
> Thanks, I think I'll pass on that one until I get my hood built ;)
Probably a good call, there. :)
Ben Gadoua
http://www.acsu.buffalo.edu/~chunglee/
Ben
Meredith L. Patterson
> If you have a centrifuge, you can buy plasmid purifcation kits from my boss.
Are these for E. coli or will they work on Gram-positive bugs as well?
--mlp
Ben Gadoua
It should work any any bacteria that can be lysed by alkaline. There are other lysing methods though, G-positive bacteria should be able to be alkaline lysed.
Ben
Meredith L. Patterson
Lactobacilli require a pre-treatment step with lysozyme, FYI.
As I think I mentioned earlier in the thread, OWW suggests that
standard column purification can be used after lysozyme/mutanolysin,
which is great and everything, and I have a box of Zymo columns ready
to throw at the question of how well that actually works. However, I
don't expect the average high school student to be able to source
columns. In Belgium, anyone can source phenol and ether by walking
into any pharmacy and handing over a few euros. If P/E (or straight
phenol) is at all effective, then I can teach "minipreps using common
household/pharmacy items plus a Dremelfuge".
Make sense?
--mlp
Nathan McCorkle
There is also this company which seems to be just who we want to deal with:
http://www.epochbiolabs.com/minispin.asp?pageName=products
250 columns for $95... you make the buffers with their recipes, can be
tuned for DNA or RNA...
nucleic acids dont differ between organisms, as long as you can crack
open the bacteria you're fine to use these.
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Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Nathan McCorkle
do catch your drift.
Cory Tobin
http://www.springerlink.com/content/n728l0l321012330/
It's the principal ingredient in chemical paint strippers. Although
the trend lately (especially in the EU) has been to move away from
dichloromethane. You may have to read the labels to find a brand that
uses it. Also, it may be mixed with other chemicals but it may still
work even as a mixture.
If you can find a brand called "Peel Away 2"
(http://dumondchemicals.com/html/peelaway.htm) that one uses
dichloromethane and methanol. The MSDS
(http://www.dumondchemicals.com/pdf/peel_away2.pdf) shows it being 70%
dichloromethane, 5% methanol and 25% "non-hazardous ingredients". I
make no guarantees but you could give it a try.
-Cory
Cathal Garvey
Keep us in the loop! Have you a website, by the way?
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Ben Gadoua
That is a non-columns purification method, it has fairly high yield. I had 100mL of log phase e.coli and got 2milligrams per millilitre and 15 or 20 millilitres (thats pretty decent, you might be able to get a little more with columns, but this method is very easy.) Dr. Lee doesn't care who you are, he'll sell it to you, he uses it to finance his research.
Ben
Nathan McCorkle
interested in just knowing what its doing more than anything... I
wonder if his kit counts towards my "buy local" (more geared towards
food) plan... lol, you are just a city away.
Christopher Kelty
use chelates for extraction?
according to this it works for e. coli.
http://www.springerlink.com/content/xl21j47276348g81/
ck
Cory Tobin
> use chelates for extraction?
>
> according to this it works for e. coli.
> http://www.springerlink.com/content/xl21j47276348g81/
I've never used that method but my guess is that it will be more
expensive than other DNA purification methods. My estimate is that a
single phenol-chloroform extraction costs around $0.20. A typical
silica column used in miniprep kits costs anywhere from $0.50 to $1
(and can be reused many times). The chelate columns, which are
typically used for protein purification, cost about $6 each.
-Cory
Cory Tobin
purify single stranded nucleic acids. So purifying plasmids and
genomic DNA wouldn't work.
-Cory
Marshall Louis Reaves
> Dichloromethane can be used in place of chloroform, as seen in this paper...http://www.springerlink.com/content/n728l0l321012330/
Then you can this strain of Xanthobacter to eat up your
dichloromethane waste.
http://www.springerlink.com/content/f13r3j721t172344/
Keep Belgium beautiful. :)
Nicholas J. Green
particularly non-polar pieces of protein and membrane that phenol
can't. On the surface of it, there doesn't seem to be any reason why
you couldn't use ether, but ether is more chemically reactive than
CHCl3, evaporates much more quickly and is a lot more flammable...its
for those reasons CHCl3 is used in DNA/RNA extractions. So just be
careful around flames and you should be fine...a side note here: ether
fumes are heavier than air so they tend to follow surfaces and pool in
low areas. Also, (at least in the US) ether is used to make
methamphetamine...so if you start buying a large amount of ether, the
local drug agency may develop a sudden interest in you.
I do minipreps on G(+) and G(-) bacteria for DNA and RNA almost daily
and can offer more advice if you need it. Best,
Nick
Nathan McCorkle
<nicholas...@gmail.com> wrote:
> As I understand it, the role of chloroform is to help dissolve the
> particularly non-polar pieces of protein and membrane that phenol
> can't. On the surface of it, there doesn't seem to be any reason why
> you couldn't use ether, but ether is more chemically reactive than
> CHCl3, evaporates much more quickly and is a lot more flammable...its
> for those reasons CHCl3 is used in DNA/RNA extractions. So just be
> careful around flames and you should be fine...a side note here: ether
> fumes are heavier than air so they tend to follow surfaces and pool in
> low areas. Also, (at least in the US) ether is used to make
> methamphetamine...so if you start buying a large amount of ether, the
> local drug agency may develop a sudden interest in you.
I've done the pheno/chloroform extraction a few times recently, and
will be doing it more for the rest of this quarter. I asked my Prof
about how it works, and remebering this thread here's what I've
compiled.
http://en.wikipedia.org/wiki/Phenol
http://openwetware.org/wiki/Phenol
It seems that what my Prof said correlates well with the openwetware
wiki... that phenol degrades proteins and membranes and solvates them,
while its also heavier than a lot of water-based buffer solutions, so
centrifugation will allow them to be separated. (meaning buffered
phenol alone should work for extractions,just might not be as clean as
if you added non-polar solvent too)
The pH of the water its saturated with moderates its charge though,
thus tweaking the solubility of other charged species, like DNA and
RNA. Phenol saturated with Tris buffer at pH 7.6ish will not dissolve
DNA or RNA, while if its soaked with pH 4.5 is will dissolve DNA but
not RNA.
If the solution you wish to purify is really dirty with
protein/membrane garbage, or maybe your samples vary in salt
concentration so the aqueous layer interchanges with the phenol layer
post-centrifugation... you then add a non-polar solvent.
Chloroform is much less polar than water, so it tends to hang out with
the phenol that also has a pretty non-polar characteristic, and thus
makes the layer heavier and aides in cleaning up the non-polar
protein/membrane crap.
Also the wiki articles say that the oxidized phenol is bad, so make
this stuff up relatively fresh.
Finally, if oxidized phenol is bad, I would stay away from non-polar
chloroform substitutes that have carbonyls or ketones.
P.S. The DCM substitution paper looks like DCM may even be better than
chloroform...
http://www.scribd.com/doc/51084375/Using-DCM-Instead-of-Chloroform
-Nathan
>
> I do minipreps on G(+) and G(-) bacteria for DNA and RNA almost daily
> and can offer more advice if you need it. Best,
> Nick
>
Cathal Garvey
a guide to amateur biotech, so it's sort of an aspiration of mine to
devise an alternative procedure. I lack the chemistry smarts by
myself, but a few promising alternatives I'd like to try include
limonene, acetone, white spirits, and any number of "essential oils"
that are cautioned against topical application (implying that they act
as solvents of biological material).
For a basic plasmid prep, you shouldn't need phenol/chloroform, as
alkaline lysis is easier and removes Chromosomal matter easily. With
the correct salt, you can use an alcohol extraction to remove RNA,
too. Indeed, I currently wonder if a miniprep procedure could be
devised that uses only store-bought ingredients (plus lysozyme from
homebrewing websites)..
What phenol/chloroform is useful for (AFAIK) is purification of total
DNA or RNA, because alkaline lysis won't recover non-supercoiled DNA.
Until there's an alternative, I'm nevertheless interested to hear
whether ether works better, though I gather Ether is prone to
spontaneous explosions.
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Cathal Garvey
a guide to amateur biotech, so it's sort of an aspiration of mine to
devise an alternative procedure. I lack the chemistry smarts by
myself, but a few promising alternatives I'd like to try include
limonene, acetone, white spirits, and any number of "essential oils"
that are cautioned against topical application (implying that they act
as solvents of biological material).
For a basic plasmid prep, you shouldn't need phenol/chloroform, as
alkaline lysis is easier and removes Chromosomal matter easily. With
the correct salt, you can use an alcohol extraction to remove RNA,
too. Indeed, I currently wonder if a miniprep procedure could be
devised that uses only store-bought ingredients (plus lysozyme from
homebrewing websites)..
What phenol/chloroform is useful for (AFAIK) is purification of total
DNA or RNA, because alkaline lysis won't recover non-supercoiled DNA.
Until there's an alternative, I'm nevertheless interested to hear
whether ether works better, though I gather Ether is prone to
spontaneous explosions.
On Saturday, 19 March 2011, Nathan McCorkle <nmz...@gmail.com> wrote:
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Cathal Garvey
a guide to amateur biotech, so it's sort of an aspiration of mine to
devise an alternative procedure. I lack the chemistry smarts by
myself, but a few promising alternatives I'd like to try include
limonene, acetone, white spirits, and any number of "essential oils"
that are cautioned against topical application (implying that they act
as solvents of biological material).
For a basic plasmid prep, you shouldn't need phenol/chloroform, as
alkaline lysis is easier and removes Chromosomal matter easily. With
the correct salt, you can use an alcohol extraction to remove RNA,
too. Indeed, I currently wonder if a miniprep procedure could be
devised that uses only store-bought ingredients (plus lysozyme from
homebrewing websites)..
What phenol/chloroform is useful for (AFAIK) is purification of total
DNA or RNA, because alkaline lysis won't recover non-supercoiled DNA.
Until there's an alternative, I'm nevertheless interested to hear
whether ether works better, though I gather Ether is prone to
spontaneous explosions.
On Saturday, 19 March 2011, Nathan McCorkle <nmz...@gmail.com> wrote:
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General Oya
It was in fact a topical application of tea tree and eucalyptus essential oils to a client of mine, that originally drew my attention to Morgellons fibers. I watched in awe when a case of what was believed to be Allergic Dermatitus Eczema began to exude these little black and white squiggly lint like fibers from between the pores of the skin, in the area of "inflammation" on hands and arms.
I've been a body worker for years and this was a first for me. It would be great to see essential oils become useful as DIY tools as well, but I imagined they would be falling into the clean your labspace kind of tools as compared to a replacement for phenol. NEAT!!!
Ryan
Nathan McCorkle
> Chloroform is nasty, but phenol is worse. It really doesn't belong in
> a guide to amateur biotech, so it's sort of an aspiration of mine to
> devise an alternative procedure. I lack the chemistry smarts by
> myself, but a few promising alternatives I'd like to try include
> limonene, acetone, white spirits, and any number of "essential oils"
> that are cautioned against topical application (implying that they act
> as solvents of biological material).
I think you need to substitute phenol with something similar....
wikipedia says limonene oxidises into carveol, which is spearmint
oil... so it could be a good thing to try:
http://en.wikipedia.org/wiki/Carveol
>
> For a basic plasmid prep, you shouldn't need phenol/chloroform, as
> alkaline lysis is easier and removes Chromosomal matter easily. With
> the correct salt, you can use an alcohol extraction to remove RNA,
> too. Indeed, I currently wonder if a miniprep procedure could be
> devised that uses only store-bought ingredients (plus lysozyme from
> homebrewing websites)..
>
I think you should more clearly define "basic", as the reason I've
heard to use phenol is to denature proteins, especially ones that can
interfere with downstream reactions like restriction and ligation. Do
you mean just getting a plasmid and then electrophoresing to confirm
its existence? If so the electrophoresis will effectively clean up the
plasmid, but while safer it seems like more work than the
phenol/chloroform extract.
I wonder if centrifuging phenol throat spray would be enough to purify
the percent or two of phenol, (enough) to use it for a phenol
extraction...
Cathal Garvey
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