Dear colleagues,
I'm currently running some in-vitro transcription assays and I also want to check the quality on a denaturing gel-electrophoresis. However, though I have a sufficient concentration outcome, I couldn't verify the quality on a gel. Currently I'm using 8,3 M Urea-polyacrylamide gels (10-6 %), running on 300 V for approx 30 min. But failed to detect my mRNAs (720-780 nt length). Do you use different systems for the detection of mRNAs?
I heard that some colleagues are using agarose gels and add bleach to denature the secondary structures. Can someone recommend this? Or would you suggest different settings regarding the polyacrylamide gel? Probably lower Acrylamid concentration and less volt?
If you just want to check the quality/integrity of the in-vitro transcribed RNA you can use a 1.2% agarose gel that has ethidium bromide (or some other dye that will allow you to visualize the RNA) in it. Just do the following:
1. Take an aliquot of your in-vitro transcribed RNA and boil it (100 degrees C) for 5 minutes in an Eppendorf tube.
2. Add loading dye (blue juice) to the boiled RNA.
3. Immediately load the agarose gel while the current is running through the gel.
4. Run the gel at ~100V until the lead dye (bromophenol blue) is about 3/4 of the way down the gel.
5. Visualize the gel to see your RNA.
Between the boiling and current flowing, it will keep the RNA denatured. I have done this for years and it never fails.
Good Luck!
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