I am totally confused about the RNA agarose gel. I have isolated total RNA from S. aureus. I want to see it in agarose gel. I ran 1% gel with formaldehyde and added 1 ug/ml EtBr during casting. But band were so so faint, can't click picture. RNA quantity I loaded was near 5 ug. Some researcher have opinion of post staining. Some says it should be added in sample. Some says it is to be added in gel.
I need a clear opinion of how much in 50 ml gel or in sample or post staining ?
Dear Dr Singh
We use EthBr in sample loading dye. It gives very good resolution. please follw the method:
http://www.its.caltech.edu/~mirsky/labmanual/pdf/rnagel.pdf
SD
Hi Samsher,
We use a short time of high voltage (200V, ~10min) to run a RNA gel, and other conditions are same with the DNA gel. Before loading, the samples were mixed with 2x RNA loading buffer, then heated for 10 min at 70 degree and chilled on ice for 3 min (according to: https://tools.thermofisher.com/content/sfs/manuals/MAN0012756_Preparation_RNA_Samples_Electrophoresis_UG.pdf).
You may load a RNA ladder to check your operation.
Best,
Xiangchun
1. RNA tends to stain poorly compared to DNA.
2. Either post-stain the gel after running the electrophoresis or mix the EtBr in sample, not mix it with the gel. The former has the advantage of removing formaldehyde while you stain and destain the gel, which is good for your next step, assuming you may transfer the RNA to a membrane.
Post staining will always be the most sensitive, but the other methods work fine if you have good signal. It sounds like you do not though.
I have success running 1% agarose RNA gels without the need for formaldehyde, and with the alternative DNA/RNA stain GelRed which can be added to the gel during preparation. The stock is usually 10,000x (and used at 1x in gel), although I find I can use 0.1x with no compromise in detection. It is much more sensitive than EtBr, and if you do prefer to post-stain you may also do so (3x final in water)
Bhai Samsher...Still you are working on this mixing of quantities...good to see...May God give you the result more faster than you are expecting....Best Of Luck and happy to know ur progress..
1. Mix 1 volume of the 2X RNA Loading Dye and 1 volume of the RNA sample.
2. Heat at 70°C for 10 min.
3. Chill on ice for 3 minutes and spin down prior to loading on a gel.
Note
RNA samples prepared as described above are not suitable for glyoxal/DMSO agarose gel electrophoresis
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