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Formaldehyde, hybridization, and Northern blots

kramerse at wmvx.lvs.dupont.com kramerse at wmvx.lvs.dupont.com
Mon Jul 18 09:28:59 EST 1994


In article <Ct37I6.G4y at acsu.buffalo.edu>, camhuber at ubvms.cc.buffalo.edu (Joel Huberman) writes:
>Dear Netters,
>
>	To size-fractionate RNA prior to Northern blotting, formaldehyde gels
>are frequently used.  Formaldehyde reacts covalently with the bases in single-
>stranded nucleic acids, preventing the bases from Watson-Crick base pairing.
>By preventing base pairing, formaldehyde prevents formation of secondary struc-
>ture, so RNAs run according to their size in the gel.  It seems to me, however,
>that the formaldehyde-modified RNA should not be able to hybridize with a probe
>after it has been Northern-blotted to a membrane.  Obviously, it does hybri-
>dize;  otherwise formaldehyde gels would not be used for Northern blottings.
>Thus, the formaldehyde reaction must be reversible.  I would like to know whe-
>ther the formaldehyde reaction is fully reversed under standard hybridization
>conditions, or is there a possibility that use of a formaldehyde gel leads to
>less efficient hybridization than would use of a standard gel or a different
>type of denaturing gel, such as a formamide gel?
>
>	If you can help to answer this question, please post your answer to the
>news group and also send me e-mail at huberman at acsu.buffalo.edu.  Thanks very
>much.
>
>Joel A. Huberman
>Dept. of Molecular & Cellular Biology
>Roswell Park Cancer Institute
>Buffalo, NY  14263
>huberman at acsu.buffalo.edu  
The formaldehyde reaction is not fully reversible under standard
hybridization conditions.  It is recommended that you bake the 
membrane to reverse the reaction for blots where low signal or
high background may be a problem. For safety I would suggest 
that you use a vacuum oven with a cold trap to collect any
formaldehyde fumes.



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