EtBr staining of RNA

Andre Hamel hamel at cc.umanitoba.ca
Mon Aug 21 13:47:40 EST 1995


In <40vpcf$5rn at hippo.shef.ac.uk> mb935645 at silver.shef.ac.uk (D L Clarke) writes:

Over the past 12 years, BRL Focus has discussed this very topic almost 
exhaustively.  What works is following:

incubate your RNA in loading buffer for final concentrations of 50% 
formamide, 3-5% formaldehyde, 0.05% dyes (BPB), 0.1 mM EDTA and 1 ug 
ethidium bromide PER SAMPLE ... heat RNA in this solution for 5 minutes 
at 65*C ... load on gel (standard formaldehyde-MOPS agarose gels 
containing NO EtBr) ... run gel ... UV-photo ... this gives optimal 
staining since RNA is soooo difficult to stain ... check past issues of 
BRL-Focus ... staining gel after electrophoresis is NOT as efficient as 
before loading gel ... the higher EtBr conc is required ... the excess 
EtBr simply migrates away in the opposite direction that the nucl acids 
migrate, thus, background fluorescence due to excess EtBr is not a 
problem compared to staining the gel AFTER electrophoresis. ... you 
should be able to see very nice rRNA banding.

all best,
Andre


		------------------------------------
     Andre Hamel                   email: hamel at cc.umanitoba.ca
     (Scientist)        tel: 204/945-7630     FAX: 204/945-8062
  Virology Laboratory,  Animal Health Centre
  Manitoba Government  Department of Agriculture
  545 University Crescent,  Winnipeg, Manitoba,  CANADA   R3T 5S6

>I have ran a 2% w/v  formamide gel for a Northern blot.
>However, when I stained the markers in 1ug/ml EtBr I could see
>nothing after a 5min incubation. I know I could have degraded
>my RNA but I would expect to see some sort of smear even
>if it has degraded.  Is single stranded impossible to visualise
>with EtBr?  Should I incubate for longer or change from
>DEPC water to TAE or TBE during the incubation.
>Any suggestions would be gratefully accepted
>Thanks
>Dave Clarke (D.L.Clarke at sheffield.ac.uk) 




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