RNA electrophoresis problem - RNA not denatured (Aug/03/2005 )
Help! I have tried running RNA extracted from brain multiple times using a standard formaldehyde gel protocol. I am denaturing the RNA for 15min at 65 then running at 100V for 2.5 hours. When I take a picture, the only thing I see is an abundance of staining at the wells and some smearing of the RNA in the gel but it looks like most of the RNA is stuck in the wells. I have tried remaking all solutions and am kind of at my wits end. any suggestions?
Hi!
Why don't you try to denaturing your RNA only 5 minutes and then run in a formaldehid gel. First run at 90 V 30 min and later at 70 V (4 or 5 h), but no at a higher V because it can degradete your RNA. I know rhis is a slow process but then you can be sure that your RNA don't be degradeted.
I'll hope this can help you!!! 
Babs
Why don't you try to denaturing your RNA only 5 minutes and then run in a formaldehid gel. First run at 90 V 30 min and later at 70 V (4 or 5 h), but no at a higher V because it can degradete your RNA. I know rhis is a slow process but then you can be sure that your RNA don't be degradeted.
I'll hope this can help you!!!
Babs
Hi
I experienced the same case as you, I suggest you repeat gel-running several times, you will find the picture you dream will appear. Sometimes, the good image are often determined by the quality of your gel-running.
Good luck
Zero
See if your formamide is oxidized. Oxidized formamide can degraded your RNA. Otherwise, run your RNA undenatured in autoclaved 0.5 X TBE for 15 min to see wheather it is degraded.
How did you purify the RNA? It sounds like there could still be proteins present which would cause the sample to stick in the well. If it was not properly denatured, it would just appear as a smear but not stick in the well
hi
for denaturing process i heat 5' at 65° and chill on ice for others 5', in order to cool the sample without enhancing renaturation process...
for denaturing process i heat 5' at 65° and chill on ice for others 5', in order to cool the sample without enhancing renaturation process...
Hi,
Do you know if this works as well as glyoxal reactions? That's currently what I use, but if I can avoid using glyoxal it will save me money for more important things.
-Hank
hi
actually, i've never used glyoxal. So i can't tell you properly for your exp. When do you use it?
Help! I have tried running RNA extracted from brain multiple times using a standard formaldehyde gel protocol. I am denaturing the RNA for 15min at 65 then running at 100V for 2.5 hours. When I take a picture, the only thing I see is an abundance of staining at the wells and some smearing of the RNA in the gel but it looks like most of the RNA is stuck in the wells. I have tried remaking all solutions and am kind of at my wits end. any suggestions?
Hi pauls075,
I extracted RNA from brain too and run it on agarose gel!!.May be it is not a good idea but it works for me!!:)Let me tell you: I prepare agarose gel with TAE like preparing DNA gel ( only I autoclave TAE before using )and also put EtBr.Then I prepare my sample for loading. for example I put 5 ul RNA +1 ul 6X loading dye ( this is the dye that I use for DNA sample loading
) + 6 ul formamide.Total 12 ul.I do not denature my RNA before loading and directly load it.Then run at 70 V for 35 min. and visualize it Previously I tried formaldehyde gel with EtBr and all gel was stained and shining!!so I could not see any band on it!!!and I decided to try this way...
While I am writing these I feel very bad:):)So you don't have to do this:)
This is due to the formaldehyde. Pop your gel into a tank of water for 10 minutes to destain and it will be fine