pH of water; dissolving RNA

Curt Ashendel ashendel at aclcb.purdue.edu
Wed Feb 19 09:16:58 EST 1997


On 18 Feb 1997 16:16:45 -0800, "Hildur V. Colot"  
<hildur.v.colot at DARTMOUTH.EDU > posted to bionet.molbio.methds-reagnts:

>Can someone enlighten me on the pH to be expected of, say. Milli-Q
>water?  I'm in a new lab and I swear that RNA pellets are not dissolving
>as well as they used to.  The pH of the water (after autoclaving) is 6.3
>on a meter and from 5 to 6 on different pH strips.  Is it a really bad
>idea to dissolve the RNA in TE at pH 7.5 before running it on a
>formaldehyde gel?  I will also refrain from drying it in a Speedvac,
>something I used to do without any problem.

I can't resist. This is one of my favorite  newbie questions.

Pure water has a pH of 7.0, plain and simple. Nothing about 
water purification changes that. Failure of meters to detect pH 7.0 is 
due to the lack of buffering capacity of pure water. That means water 
has a difficult time affecting the sensor of the free hydrogen ions 
without it being affected itself. Meters do better than paper, which 
does not turn at all with water and simply looks like wet pH paper. 
For all the pH paper I have ever seen, that color is at the extreme 
acid end of the range.  Believe me, your RNA pellet should go into 
water as well as TE, unless the RNA was precipitated with acid or 
contains denatured protein. RNA just takes a while to dissolve. Be 
patient, and be careful, since it is during this dissolution that 
RNases do a lot of their dirty work (sorry, but unless you kept 'em 
out of your RNA prep, there is little you can do about them at this 
point).

This reminds me of a story about water and pH paper. At some time 
in my past my lab's aqueous radiological waste was mostly unbuffered 
water from washes of P81 paper from kinase assays. They require that 
it be neutral pH before pickup for handling safety reasons (some labs 
used to use a lot of TCA precipitations that made the waste noxiously 
caustic, so they require neutralization before pickup). They check the 
pH with pH paper and used to tell us that our waste was too acidic to 
pick up. I had to explain to them that it was nearly pure water, and 
there was no acid in it at all, and then I gave them my mini-lecture 
on pH paper not registering pH with highly dilute buffers. For a 
while they did not believe me and we had to dump in some 1M pH 7.0 
phospate buffer to get the waste to register on pH paper.


 

Curt Ashendel
Purdue University, West Lafayette, IN
ashendel at purdue.edu



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