I had gel-purified the RNA band of size 240nt from in vitro transcription reaction. But now i can see that one extra band is coming at top. RNA samples were denatured for 10 min at 80 degree Celsius and immediately placed in ice before loading. And this is happening multiple times with independent preps. First two lanes of the attached image.Thank you
Hi,
Why did you put denatured RNA on ice before you load it? Do you know that after denaturing if you put RNA on ice it will form the secondary structure and may bind to something on your solution (read refolding RNA aptamer).
ideally RNA samples should be loaded immediately after denaturing it..putting on ice prevents immediate re folding of RNA but whn u load on gel it get gradually heated till it reaches RT. probably tht is why u see the top band.
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