cannot see RNA on gel - (Aug/25/2006 )
I am attempting to run a Northern using total RNA. I bought this all-inclusive (sort-of) kit from Ambion to run the assay. After running 30ug of total RNA on the denaturing gel, I stained the gel for 20min using Ethidium Bromide. I didn't see anything!! The kit directions say to stain the gel in ethidium diluted with the 1x running buffer. Weirdly, after transferring the RNA to the membrane, I stained the membrane using methylene blue and lo and behold, I saw the 23s and 18s rRNA bands. Any ideas on what to do? I tried to run a gel today to assess my RNA quality using 5ug and again saw nothing! Am totally baffled.
is your UV bulb burnt out, or burning out?
is your EthBr good? heat and light can degrade it...how is it being stored and have you used it for anything else lately to check it?
almost has to be a problem with the staining or visualizing; it seems as though your RNA is probably there
that's the weird thing. . .we use this EtBr almost everyday to stain DNA gels, so I know that both the EtBr and the UV bulb are good. do you need a higher concentration to see RNA vs DNA? I'm stumped.
i put 1µl of a 0.4mg/ml EtBr solution in the sample for agarose gels
for PAGE, i stain for 15' in 0.5µg/ml EtBr solution (Generally 0.5XTBE supplemented with EtBr)
does it matter that I tried to stain the gel after I ran it-as opposed to adding the EtBr directly to the samples?
honnestly i don't know. I suppose if the gel is thin both methods should be fine.
You may consider that and test on few samples.
thanks! I am running my Northern tomorrow and plan on cutting off the lane that contains the size marker and staining it separately. Will see if its visible!