RNA extraction

GLYCOBIOLOGISTS OF BUFFALO camlau2 at ubvms.cc.buffalo.edu
Fri Nov 5 12:20:00 EST 1993


In article <2bc89b$t2e at nntp.ucs.ubc.ca>, sarven <sarven at ecc.ubc.ca> writes...
>In article <2b9odo$ojq at nntp.ucs.ubc.ca> sarven, sarven at ecc.ubc.ca (who is
>me) writes:
> 
>>.  As for electrophoresis I like
>>formaldehyde gels.  Here is my protocol for running an 80 ml gel:    
>>    1.) .8g agarose (1%gel) is melted in 68 mls of DEPC water.
>>    2.) After the gel is cooled to about 55 degrees celsius add 4 ml of
>>formaldehyde and 8ml of 10X MOPS (.2M MOPS, 80 mM sodium Acetate adjust
>>pH to 7 and then add10mM EDTA ph 8) and 1ul of 10mg/ml ethidium bromide.
>>    3.) Run the gel in 1X MOPS slowly overnight (Then you don't have to
>>worry about washing off ethidium bromide) 
  ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
	Why are we supposed to be worrying about that?  

>>Also, before I run the samples, I heat them at 65 degrees celsius in
>>loading buffer for 15 minutes and load them immediately onto gel (NOTE:
>>don't put the samples on ice after heating).
  ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
	Why??  I used to do this method all the time, and I always put my
samples on ice after heating and before loading, and I never worried about the 
EtBr (which was in my samples, not in the gel) 
The procedure worked fine for me, except that I have since found that I MUCH
prefer glyoxal denaturation on TAE gels for band sharpness, olfactory benefits, 
and the ability to run gels of a lower percentage because the gel is a native 
one and there's no nasty formaldehyde weakening it.

					-=Annette=-



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