DNA dissolution problems
Jonathan Nesser
have to do my protocol within a certain time period (this is the high
school demonstration again), so I don't have the time to wait for
several days for the DNA to dissolve if this happens when I'm doing my
full protocol. My protocol calls for ethanol precipitation twice, and
I'm using a centrifuge to do so. Today, after I got the DNA pellet-
ized, it wouldn't go back into solution. I'm not sure if I centrifuged
it too long, let it dry too long (only sat with no solvent in a closed
micro centrifuge tube for ~20 mins), or what, but I tried both using
pH 8.0 tris buffer and heating at 60C for ~1 hr, and neither seemed to
have much effect. After doing both, I did notice some dissolving had
taken place, but the bulk of the pellet was either still a chunk or
had just broken down into smaller chunks (just as likely from my
repeated finger-flick stirring as from any dissolving).
I read that precipitation pellets which refuse to dissolve are most
likely RNA on some forum, but I'm not sure how reliable that
information is, as I haven't heard it before. I haven't really had
this problem before today, but it would be very inconvenient if it
happened during my demonstration. Even if there is no way to make
pellets dissolve faster, does anybody have any ideas as to what caused
this problem in the first place? Would over-centrifuging (I've just
been centrifuging until I see obvious and solid looking pellets, could
I have overlooked them today and taken it too far)? Also, I read on a
forum that people were doing ethanol precipitation with 70% ethanol,
I'm using pure lab grade 90%+, would that have anything to do with it?
Anything else I'm not thinking of?
Sorry for the vague questions, but I tried every trick I could find in
the literature besides .8mM NaOH (the step after each precipitation
requires a specific pH) and couldn't get anything to work, and I can't
really afford for this to happen when I do this demonstration. Thanks
in advance for your time.
Jonathan Nesser
jonatha...@gmail.com
diybioandneurosci.blogspot.com
Nathan McCorkle
EtOH... the water helps dissolve DNA for sure
I doubt its RNA, but again try more water. I usually dry my DNA with
the microtube open, or even in an oven for a while sometimes to dry it
sufficiently, I've done midipreps and maxipreps, and never had
dissolution problems (though that's mainly plasmid DNA). What kind of
DNA are you expecting, genomic or sheared?
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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Cathal Garvey
When you dry a pellet that's wet with 70% EtOH, the EtOH dries quickly
and the residual moisture is mostly water. When you dry a pellet of
90/99% EtOH, there's not much moisture left at all.
I have heard that this issue involves denaturing of the DNA in its
dry-state, forming a "clot" similar to the sort that forms during a
miniprep. In other words, the DNA strands are tightly packed and
cross-linked. If this is so, then some heat may help them to redissolve,
but you'll have to be careful not to burn the DNA: outside of solution
it may be more susceptible to mild or serious damage from heat.
Perhaps step it up to 60C for a while to see does that help it dissolve,
and if that fails try 80C, then 95C.
If you care about the DNA result, repeat the extraction rather than
leaving it at 95C for an hour; depurination (decay of one nucleotide to
another) can occur at extended high temperature incubations.
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jlund256
smaller or fragmentary as the DNA dissolves out and leaves the gunk
behind. These quick demo protocols often leave cell debris. Being
conservative--leaving some supernatant after spinning down cell debris
may help. Longer spins, faster spins, longer protease digestion, and
extraction with phenol:chloroform also help.
Jim
Jonathan Nesser
Thanks for the replies everybody, this gives me some directions to go
in next time I run into this problem. I'll start using 70% etOH during
extractions ( side question, will that reduce yield, and should i let
the etOH sit longer to precipitate?), as well as spin down the cell
bodies longer during that step. If I wind up with one of these clots
again how bad would it be to poke it a few times with a micropipette
tip to break it up (in theory giving whatever dna is caught in the
cell body clot more of a chance of being exposed to the solvent and
dissolving), then pipetting the solution out while avoiding the cell
clumps, or even recentrifuging to spin them back to the bottom?
Jonathan Nesser
jonatha...@gmail.com
diybioandneurosci.blogspot.com
Nathan McCorkle
terms of quality and utility. Its sucks off fatty cell junk and
hydrophobic crap, deactivates most enzymes (DNAse, some RNAse), and is
pretty easy overall.
We've talked about alternatives if you can't get phenol and
chloroform, but never reached an agreement on replacements:
http://groups.google.com/group/diybio/browse_thread/thread/da735df04bfc4067
70% over 90% EtOH shouldn't change yield, but the 70% will have more
water to ensure that the DNA doesn't get too dry during the drying
step.
I love the third reference in this wikipedia article, it gives real
data for the different temperature and time conditions for an ethanol
precipitation/extraction... I don't chill on ice anymore, and my
yields are generally better than my classmates (though that could be
due to better technique in other areas)
http://en.wikipedia.org/wiki/Ethanol_precipitation
This is the 3rd reference in that wiki article.
The tables and graphs are of interest here:
http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.34900.File.dat/Focus%20Voume%209%20Issue%202.pdf
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Cathal Garvey
buffer to yield a less-than-100% final mix. You *could* use 70%, but
you'll have to add a lot more to achieve the precipitating concentration
of EtOH you need. But, for washing, using 70% couldn't hurt; it'll
prevent you from drying out the pellet completely, which can lead
(apparently!) to difficulty re-dissolving pellets.
Although, as someone else mentioned, it's possible that the residual
pellet isn't entirely DNA, also.
As to Phenol-Chloroform, bear in mind the toxicity of both ingredients
when considering whether to use it for a lab demo. Phenol can cause
burns, and is even stronger when mixed with Chloroform.
--
Jonathan Nesser
phenol/chloroform, so that is a moot point for this instance - and
with those safety issues I probably wouldn't risk exposing it to a
bunch of lab technique lacking high school students, though it sounds
interesting for my personal work. That article was definitely
informative, though it follows what I've already been doing - mixing
the cold ethanol with room temperature solution and letting "incubate"
at room temperature for several minutes. I guess sometimes it's nice
to not have been exposed to all those old lab dogmas that have never
been tested. Also, I hadn't heard about using ammonium acetate to
remove protein before (I'm sure it's common in the literature, just
haven't been reading preps that were that complex up to now - too
focused on getting my simple prep working).
On Jan 20, 12:52 pm, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Use 100% EtOH for first precipitation, because it'll be mixed with the
> buffer to yield a less-than-100% final mix. You *could* use 70%, but
> you'll have to add a lot more to achieve the precipitating concentration
> of EtOH you need. But, for washing, using 70% couldn't hurt; it'll
> prevent you from drying out the pellet completely, which can lead
> (apparently!) to difficulty re-dissolving pellets.
>
> Although, as someone else mentioned, it's possible that the residual
> pellet isn't entirely DNA, also.
>
> As to Phenol-Chloroform, bear in mind the toxicity of both ingredients
> when considering whether to use it for a lab demo. Phenol can cause
> burns, and is even stronger when mixed with Chloroform.
>
> On 20/01/12 18:48, Nathan McCorkle wrote:
>
>
>
>
>
> > Phenol:chloroform extractions are easy and worth a million bucks in
> > terms of quality and utility. Its sucks off fatty cell junk and
> > hydrophobic crap, deactivates most enzymes (DNAse, some RNAse), and is
> > pretty easy overall.
>
> > We've talked about alternatives if you can't get phenol and
> > chloroform, but never reached an agreement on replacements:
> > 70% over 90% EtOH shouldn't change yield, but the 70% will have more
> > water to ensure that the DNA doesn't get too dry during the drying
> > step.
>
> > I love the third reference in this wikipedia article, it gives real
> > data for the different temperature and time conditions for an ethanol
> > precipitation/extraction... I don't chill on ice anymore, and my
> > yields are generally better than my classmates (though that could be
> > due to better technique in other areas)
>
> >http://en.wikipedia.org/wiki/Ethanol_precipitation
>
> > This is the 3rd reference in that wiki article.
> > The tables and graphs are of interest here:
> > On Fri, Jan 20, 2012 at 1:33 PM, Jonathan Nesser
> > <jonathan.nes...@gmail.com> wrote:
>
> >> Thanks for the replies everybody, this gives me some directions to go
> >> in next time I run into this problem. I'll start using 70% etOH during
> >> extractions ( side question, will that reduce yield, and should i let
> >> the etOH sit longer to precipitate?), as well as spin down the cell
> >> bodies longer during that step. If I wind up with one of these clots
> >> again how bad would it be to poke it a few times with a micropipette
> >> tip to break it up (in theory giving whatever dna is caught in the
> >> cell body clot more of a chance of being exposed to the solvent and
> >> dissolving), then pipetting the solution out while avoiding the cell
> >> clumps, or even recentrifuging to spin them back to the bottom?
>
> >> Jonathan Nesser
Nathan McCorkle
Please note my emphasis was on the incubation times and temperatures, rather than the buffer specifics.
Tou can also get cheap spin columns from Epochbiolabs
Sent from my mobile Android device, please excuse any typographical errors.