Hi everyone,
I would like to run a denaturing agarose gel to view RNA more precisely and we do not have a recipe for making such a gel in the lab. Does anyone have an easy and effective recipe for RNA? Thank you.
Gurjot
Here is some further reading:
"Denaturing RNA electrophoresis in TAE agarose gels" (Analytical Biochemistry, Volume 336, Issue 1, 1 January 2005, Pages 46–50)
and another website with protocols:
https://www.nationaldiagnostics.com/electrophoresis/article/preparation-denaturing-agarose-gels
Thank you all for your answers. I will first try formaldehyde recipe.
best regards,
gurjot
A word of warning: those formaldehyde gels are very brittle, so take care not to break them when you are handling them. It's a little bit of a pain to have to piece them back together.
We used to use old x-ray film cut-to-size to kind of scoop them up and support them, but nobody uses x-ray film anymore! On the other hand, if your gel is small, it won't be too hard to keep it from breaking.
Hi, use the formaldehyde gel, it is working very well but as David said the gel is brittle and you should handle it carefully. Good luck!
Here is the recipe for 1.5% denaturation agarose gel. To 1.5gm of agarose add 10ml of 10X formaldehyde denaturation buffer (200 mM MOPS, 50 mM sodium acetate, 10 mM EDTA adjust pH to 7.0 with NaOH) and 90ml of water. Dissolve agarose in microwave. Cool and add 1.8ml formaldehyde (37%). Mix thorougly and pour onto gel support.
Use 1x Formaldehyde denaturation buffer for electrophoresis. After completion of electrophoresis, stain with ethidium bromide.
Your answers helped me as well, thank you!
If I may ask a follow up question: How important is it to use DEPC treated water to prepare all the buffers for the gel?
The main thing is that you want to be sure you have RNAse-free water. DEPC treatment will inactivate nucleases, so that is the standard way to ensure "RNAse free" water. I have used water out of a well-maintained MilliQ system for a quick RNA gel and had no troubles, but DEPC treatment is not difficult and probably worth the effort. If you get RNAse contamination, your RNA will be compromised.
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