Does anyone has a denaturing RNA gel protocol, by just adding guanidine to the sample, and running in a regular agarose gel?
Hi,
The following gel electrophoresis conditions are recommended:
- use 1X TAE buffer instead of 1X TBE
- use agarose gel in the concentration of 1.1%-1.2%
- add ethidium bromide (EtBr) to the gel and electrophoresis buffer to avoid the additional (potentially RNAse-prone) step of gel staining
- always use fresh gel and buffer as well as clean electrophoresis equipment for RNA analysis. Wear gloves to protect RNA samples from degradation by nucleases and avoid a hand contact with EtBr. Do not use high voltage to avoid RNA degradation during electrophoresis.
2. Heat an aliquot of the RNA solution at 70°C for 1 min and place it on ice before loading on a gel. Best wishes :)
I agree with all of the above: you don't need a denaturing gel, you just need to ensure the RNA is denatured before loading. If you want to really make sure, the only modification I would suggest to the above advice is to make up your loading buffer to a final conc. of 60-75% formamide.
So:
6x loading buffer: 2ul
RNA solution: 1ul
Formamide: 9ul
Heat this to 65 degrees for 5min (or 70 for 1 min: 65 is essentially the highest you can take RNA before it starts degrading), then crash cool on ice as suggested. The heating/crash cooling denatures the 2ndary structure and prevents reannealing, and the formamide acts as a chaotropic agent to prevent reformation of 2ndary structures while you're loading/running the gel.
We use a bleach gel. They are simple, inexpensive comparably and effective. Here is a link to an article https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699176/. Message me if you want more detailed recipes or protocols and I will send them your way when I am at work. Good luck!
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