Help: Can't see biotin:riboprobes in Agarose gels

James Drummond drummond at ncifcrf.gov
Thu Sep 28 12:04:28 EST 1995


In article <mabman-2808952038100001 at mabman.vip.best.com> mabman at best.com (J S Abrams) writes:
>We are trying to characterize biotin labeled riboprobes for in-situ
>hybridization. We've been generating our probes using the Gibco labeling
>system. Currently, we are trying to run these on a 1% agarose gel containing
>approximately 2.2M formaldehyde. We've been following Maniatis as far as gel
>running buffer (.02M MOPS), gel sample buffer and loading buffer. After
>running the gel, we rinse it in several changes of water and stain it with
>5ug/ml of EtBr and attempt to destain. We haven't been able to stain these
>successfully and have heard many conflicting  theories about EtBr being able
>to stain RNA. Any opinions on how to optimize the staining  of formaldehyde
>gels? How long to wash out the formaldehyde?  Also, if EtBr only
>intercalates into double stranded nucleic acid how does this technique
>work?  If it requires the RNA in the gel to have secondary structure (i.e.
>hairpin turns/loops), how universally applicable can EtBr staining of
>riboprobes be, since it would tend to be sequence specific, right?
>
>Thanks for your help and advice.
>
>John S. Abrams, PhD
>DNAX Research Institute
>Palo Alto, CA
>John_Abrams at dnax.org

Try staining the RNA in the loading buffer.  Then load it on the gel and run
it.  This will save you all the rinsing and destaining.  EtBr does stain RNA in
a denaturing gel.  I have done it many times.  Try running a few commercial
standards in your gel to make sure that one of your buffers is not contaminiatedwith Rnase.  This is a problem with RNA.  Be sure that all tubes and pipettes
that might contact your RNA are also Rnase free.

Good luck

James Drummond
Drummond at avpvx1.ncifcrf.gov




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