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

J S Abrams mabman at best.com
Mon Aug 28 15:38:10 EST 1995


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



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