After cutting the band out of the 8M Urea gel, what should I do to elute the RNA? Which elution buffer should I use and how to clean up the salt? Is phenol-chloroform extraction a necessary step for desalting?
Thanks a lot!
You can use ZR small-RNA™ PAGE Recovery Kit . To know about its protocol visit http://www.zymoresearch.com/downloads/dl/file/id/155/r1070i.pdf
We just use the Ambion Spin columns to get rid of the polyacrylamide, but for the rest you can do elution of RNA from a page gel without any kit.
- cut out your desired fragment from your PAGE gel
- Spin it down through an ep (with punctured holes in the bottom) inside a new ep. With higher concentrations of acrylamide you might want to pre-cut your gel.
- Add 400uL 0.3M NaCl to the slush and rotate overnight at 4 degrees
Next day:
- Transfer slush (liquid + remainder acrylamide) to Ambion spin column (AM10065)
- spin down
- Perform an Ethanol-precipitation on your RNA (you might want to add glycogen to low amounts of RNA)
I've used this very simple protocol with great succes hundreds of times. I generally recover around 50% of my RNA:
- Transfer your gel slice to a tube containing 200-400 µl 0.3M NaOAc, pH 5 and incubate overnigt at 4 degrees with rotation. If you are afraid of RNAses add an equal volume of Phenol:Chloroform:Isoamyl Alcohol 25:24:1.
- Spin down and transfer aquas phase to a new tube.
- Precipitate with 2-3 vol. ethanol.
That's it! If you are in a hurry you can incubate at room temperature for a couple of hours with similar results :-)
Thank you so much Alex and Mikkel! I have one more question. I noticed that some people use 1xTBE or TE for the O/N elution and a Cold spring harbor protocol specializes a SDS-containing elution buffer (basically NaOAc + EDTA + SDS). As I need to further label my recovered RNA with biotin at the 3'-end, I am wondering which elution buffer, followed by ethanol precipitation, would minimize the salt and also urea contamination that may inhibit the T4 RNA ligase.
In principle, you can elute in all buffers, since the method relies on simple diffusion of the RNA into the buffer - even water should work. I would think that they add SDS to denature the gel and the proteins in the gel to optimize RNA relase, but I'm not sure. Adding PCI might also improve the diffusion....
I never have problems with salts or Urea when I elute in NaOAc. I also use my RNA in downstream reactions - ligations etc. No problem :-) Off course remember to wash your RNA pellet in 70-85% Ethanol after precipitation to remove salts. One benefit of the protocol that I use, is that you don't smash up the gel into tiny fragments and therby prevent too much Urea and acrylamide contamination.
Hi Liu, i follow crush and soak method with CSH recommended buffer (NAoAC+ EDTA+SDS) on rotation O/N. pass through gel removing column from zymo to remove acrylamide pieces, then i purify the supernatant using RNA purification kit.
zymo is my choice
i re use the columns used for removing acrylamide several times. the yields i get using this method are fantastic.
if you are still worried about yields, you can try this method
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