DIG-labeled RNA probes

[Gae Kovalick]

I've been having some difficulties synthesizing intact DIG-labeled 
RNA probes for in situ hybridization, 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 gel.  Lately, I've 
been seeing a smear on the gel instead of a discrete band.  I've 
tried three different constructs and gotten the same results.  Each 
construct consists of a short (200-400 bp) DNA fragment inserted into 
the Bluescript vector.  All the inserts are different DNA sequences. 
I've checked the template DNA on a gel, and it seems intact.  The 
template is digested with either EcoRI or HindIII prior to RNA 
synthesis.  Before the RNA labeling reaction, I treat the template 
with proteinase K, followed by several organic extractions and an 
ethanol precipitation.  I add an RNase inhibitor to the labeling 
reaction.  I get the same results (a smear) whether I use T7 or T3 
RNA polymerase (I prepare both sense and antisense probes).  Before I 
run the probe on a gel, I clean the gel box thoroughly and use 
DEPC-treated solutions to prepare the gel and the running buffer. 
Why are my probes so crappy-looking?  Any suggestions?

Thanks,


Gae Kovalick, Ph.D.
Department of Science and Mathematics
The University of Texas of the Permian Basin
4901 E. University Blvd.
Odessa, TX 79762-0001
915-552-2265
915-552-3230 (fax)
kovalick_g at utpb.edu
www.utpb.edu/scimath/kovalick/

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