DIG-labeled RNA probes

Hong Fen hongfen at tll.org.sg
Tue Feb 7 02:29:35 EST 2006

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

Rice Functional Genomics
Temasek Life Sciences Laboratory
An affiliate of NUS and NTU
1 Research Link
National University of Singapore
Singapore 117604 
Tel : (65) 6 872 7486
Fax: (65) 6 872 7518

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