I need to load and run 5μg of total RNA per sample for posterior Northern blotting. I have ran it in an 1% LE Agarose gel overnight at 25v and obtained horrible "U" shape bands. I thought it could be a case of not letting the gel settle long enough, I ran it again in 1% and 0,8% LE agarose at +4ºC overnight at 20V making sure that the gel was optimally settled. Still got this messy bands.
Any idea of how to get sharp bands?
The thing is that in very first Northern performed, in the same conditions, the 5μg of RNA yielded clear sharp bands and never managed to get them again.
The running buffer I'm using is the one provided with the NorthernMax Gly Kit for Northern blotting.
Thanks a lot!!
I would recommend running a 1.5% high-resolution gel at 1-2V/cm voltage using an agarose LE that resolves more finely with a higher resolution ( https://bit.ly/2lULOwK ), and perhaps you may also consider preparing a fresh running buffer (though your current one may work just fine since it did so on the first run). Hope this is helpful.
Kundan Das Thank you very much! I tried changing the % of agarose, different buffers and voltages and the resolution significantly improved. I also realised that the RNA samples that were stored at -80° were more prone to present this U shape so now I dont move them from -20 unless strickly necessary for long term storage.
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