Heating Northerns Instead of Formaldehyde
ffr at inel.gov
Mon May 1 13:18:56 EST 2000
>Biotechniques just published a note where you use hot buffer instead of
>formaldehyde. It sounds less hazardous and they claimed better
>resolution and sensitivity. I wonder if anyone has experience with
>this technique, and I wonder if the formaldehyde is omitted , if the
>RNAses begin to muck things up?
We have started using the hot buffer method for Northerns,and found that
it works great. We don't get too carried away with temp control, but do
follow the protocol by starting with preheated TAE buffer (60 °C).
During the electrophoresis, we run at 100 V in a Bio-Rad gel box (10 cm
long gel), and the temperature is typically maintained between 45-50 °C.
This method still requires a small amount of formaldehyde in the sample
loading buffer, but avoids the use of formaldehyde in the running buffer.
Incidentally, we've also incorporated the downward transfer protocol in
50 mM NaOH cited in that paper (cf. Biotechniques 25:420-425, 1998), and
were very suprised at the significantly greater amount of RNA
transferred by this method (visualized with methylene blue), as opposed
to a conventional upward transfer in SSC.
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