Formaldehyde pH for Northern blot
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Formaldehyde pH for Northern blot (archive)
We've been having trouble with our RNA degrading during Northern's.
We've discovered the culprit is the formaldehyde and particularly the
denaturing step (we denatured with formaldehyde in the sample). The pH
of our formaldehyde was (4.4) which we had pH'd from protocols which
mentioned the pH should be >4. However, after running some analytical
gels we discovered that a pH of 5.6 or even 6.6 gives much better
retention of the sample and that we're degrading quite a bit at pH 4.4.
Basically, I'm wondering if this is a common problem and whether labs pH
the formaldehyde every time they run a northern, and if so, what is the
optimum pH?
Link
Hi,
a solution of formaldehyde gas in water should have a neutral pH. So the optimum
pH=7 , which can not be measured with a pH-electrode because the resistance of
the solution is too high. (There are practically no ions present) The
formaldehyde will be converted to formic acid by air oxigen. Now the,acid, pH
can be measured with a pH electrode. I don't think that neutralising the formic
acid is a good idea. The best thing to do in practice is 1) take a fresh bottle
of formalin ; should last for month if kept tight. 2) make fresh ,non-oxydised
formaldehyde, from para formaldehyde , a polymer of formaldehyde. Imo a very
nice alternative for the formaldehyde is the , reversible, chemical reaction of
RNA with glyoxal in DMSO containing buffer. It forms rings with RNA preventing
H-bonds by sterical hinderence. This reaction product is stabel. So , after the
reaction , the electrophoresis can be done on the bench in a standard buffer.
After blotting to nylon the reaction can be reversed by heating the nylon blot
at 65 pH=8 (tris/cl) for 10 min. See maniatis for details.
--
Gys
Link
> So the optimum
> pH=7 , which can not be measured with a pH-electrode because the resistance of
> the solution is too high. (There are practically no ions present)
You are kidding right????
Last I heard, pH7 meant that you had 10exp(-7)moles/L. A pH ellectrode
should have absolutely no problems whatsoever reading a pH of 7.
Also it is difficult to have a solution of gas. Either it is dissolved or
it is not.
> The formaldehyde will be converted to formic acid by air oxigen.
I do not think this is true either. I am no organic chemist but I believe
that Formaldehyde which is a gas (bp -21C) at room temperature can be
dissolved into water to form a solution. The solution that I and others
buy is a 37% aqueous solution that is often called formalin. In the
solution of Formalin, Formaldehyde reacts with water via a hydration
reaction to form Methanediol not to be confused with Methanoic acid
(formic acid). It is one of the few carbonyl compounds that exsist almost
exclusively as a hydrate in solution.
The reason that the running buffers pH will drop has more to do with
elctrochemistry. I personally have never found formaldehyde to be the
culprit of poor quality RNA but maybe I have not done enough Northerns yet
--
Peter Pediaditakis
Link
Straight formalin (35 - 37 % formaldehyde solution) will turn *bad* on prolonged
storage. Two formaldehyde molecules will react to form methanol and formic acid.
This reaction can be retarded by adding methanol (some commercial preparations have
about 10 % methanol added), or the acid can be captured (other commercial
preparations have a slurry of some acid-binding substance on the bottom). Storing in
the cold might slow the breakdown of the formaldehyde, it will also increase the
polymerization to paraformaldehyde, especially when the solution is acid.
Usually formalin is diluted into some buffer before use, which will take care of pH.
However, for very critical applications it is still best to prepare a fresh solution
from paraformaldehyde.
Regards, Henk
Link
> Imo it is of little use to measure the pH
> of a solution which has a very low electrical resistance/buffer capacity.
> So I would not use pH of formalin as a quality check.
You are most correct that it is difficult to accurately measure the pH in
low ionic strength solution but it is easy to alter the solution quickly
to get a good measurement. If you add 50 ul of 1 M KCl in milliQ water to
1 ml of your low ionic solution, you will get a accurate measurement of
the pH
--
Peter Pediaditakis
Link
Last update 22-Dec-2000, Rating Good of 0 votes.
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Formalin solution stability in terms of formic acid.
Anyone has any idea to prevent or retard the degradation of formaldehyde into formic acid, please email to the address above. Thanks in advance.
Regards,
Arif
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Hi,
I'm very glad to find like this site.
I'm going to do Northern blotting but I faced an one problem. After blotting, I check the gel to know the retained RNA on gel. There is problem which the big band appeared never expected.
Would I know this reason?
Best regards,
Hyun-Jung Ryu
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i was wondering how and why formaldehyde is used to preserve animals. How was formaldehyde ever discovered as a machanism for preservation?
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i was wondering how and why formaldehyde is used to preserve animals. How was formaldehyde ever discovered as a machanism for preservation?
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In response to degrading RNA during Northern's... First, I think that all the responses from your question go off on a tangent and do not get you any closer to an answer to your question, so... Second, Put away your electrodes. There is NO need to pH formaldehyde solution (aka formalin, 37% formaldehyde) to run a Northern!! note... You may want to pH your gel running buffer solution, but it should be alright if you make it according to protocol (if you want to know more about this email me). You say your trouble is with the denaturing step? What are you using to denature the sample? Since I don't have the answers to the latter question I will comment on that. Use RNA sample loading buffer from Sigma. You just add a specified amount of RNA sample loading buffer, heat at 65C for 10', then load your sample... Third, I do believe that the formaldehyde pH of 4.4 was helping your RNA degrade, but your RNA will be degraded almost exclusively during the RNA preparation. (Unless for example, you are using straight formaldehyde solution to denature your sample and not an appropriate denaturing/loading buffer). If you want to explore this avenue, feel free to email me.
For solid advice on this and other questions, without a forum atmosphere (which tends to give response far from what you originally asked), please visit a new site which is intended to help answer common lab problems, at http://www.labadvice.com
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