siRNA purification - (May/10/2006 )
I'd like to label siRNA with a radio tracer. Has anyone done that yet?
I have an idea and protocols, but I need to know, how I can purify the labeld siRNA from low molecular chemicals and salts. Is there a gel filtration column I could use with a 13,302 Da siRNA, 21nt?
And how small of an amount of siRNA can be detected by a UV-detector?
Thank you for helping!
T4PNK can label RNA as wel as DNA. So it may be your way to do.
For purfication : a G25 column seems ok for that purpose. But never tested it.
So i would also try a PAGE purification method in native conditions (or you can do a denaturing page, and anneal again after gel elution.
I have experienced with labeling siRNA using T4PNK as mentioned by fred,
After that purify the labeled siRNA by mini Quick Oligo columns (Roche) for subsequent hybridization work…
If your siRNA are come from in vitro, you'd better dephosphorylated them before T4PNK labeling.