DIG-labeled RNA probes

[Hong Fen]

I've been having problem with stability of DIG-labeled RNA probes, and I'm wondering if anyone can give me some suggestions on what the problem might be and how to correct it.

When I synthesize RNA probe, I always check the integrity of the probe by running an aliquot on a FA gel, but the denatured one (65 C for 5 mins) always shows very faint band compared to the same size of the undenatured one(it always show one more big band -- is it second structure?) .  

I've tried QIAquick PCR purification kit and phenol method for template prep, I'm going to try MinElute Reaction Cleanup kit to remove RNase completely and get a good yield.

I add RNase inhibitor in the transcription reaction and use RNeasy Mini Protocol for RNA Cleanup to purify the probe. I always clean the electrophorosis tank, gel tray and comb with a solution of 0.1M NaOH, 1mM EDTA pH8.

Why the band of my probe becomes weaker after being denatured?  Any suggestions?

Thanks and Best regards,
Hongfen Luan





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