Seconded, it could be the absence of water from the dried pellet.
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.
On 19/01/12 05:10, Nathan McCorkle wrote:
> It could be protein/fat contamination, or that you're not using 70%
> 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?
>
> On Wed, Jan 18, 2012 at 9:11 PM, Jonathan Nesser
> <jonathan.nesser@gmail.com> wrote:
>> I ran into a problem at the lab today concerning DNA solubility. I
>> 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
>> jonathan.nesser@gmail.com
>> diybioandneurosci.blogspot.com
>>
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>
>
>
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