>>> Help about formal/agarose ges <<<

David S. Huen smh1008 at cus.cam.ac.uk
Fri Oct 14 10:04:33 EST 1994


In article <danielp.1.2E9B4B7B at kahu.lincoln.ac.nz>,
danielp at kahu.lincoln.ac.nz (PHIL DANIEL) wrote:

> In my recent and limited experience, unless there is A LOT of RNA on the 
> gel, it is necessary to destain the gel a long time (several hours) to see 
> anything. This is because the formaldehyde and EtBr together produce high 
> background fluorescence.
> 

At the risk of repeating something that may already have been said, I find
that soaking in something with a high no. of amino groups (eg. 1M ammonium
acetate or Tris) eliminates the formaldehyde thru Schiff base formation
fairly rapidly and makes subsequent staining much easier. 

I would also point out that formaldehyde gels denature the RNA for
electrophoresis by forming Schiff bases with the nucleosides. As long as
those adducts are there, your RNA won't hybridise to your probe decently
(since that is the intention of the adduct in the first place!). Removal of
the formaldehyde adduct from the base is considerably more difficult than
the equivalent adduct with glyoxal, which has the decency to fall apart at
pH 8. Now this presumably occurs during baking when transferring to
nitrocellulose. However, some of the newer protocols (alkaline transfer etc
to modern membranes) don't seem to properly remove these adducts in my
hands such that I find soaking the membrane in hot 1M Tris or ammonium
acetate briefly (10 mins) gives me much better results. Hopefully it isn't
all in my imagination.

-- 
David S. Huen                        Phone: (0223) 333921
CRC Human Cancer Genetics Group      Fax:   (0223) 333875
Dept. of Pathology                   e-mail: smh1008 at cus.cam.ac.uk
University of Cambridge
CB2 1QP                              
United Kingdom



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