rRNA visualization in formaldehyde gels

Robert Hartley rh at mblab.gla.ac.uk
Mon Oct 23 18:52:32 EST 2000


In article <v04011702b61a0d1d29b7@[144.92.64.174]>,
mlsulliv at facstaff.wisc.edu ("Michael L. Sullivan") wrote:

> >Hi.
> >
> >I can not see any rRNA on the formaldehyde gel.
> >
> >The protocol is the one in Current Protocols : agarose gel, formaldehyde and
> >MOPS. The loading buffer is sterile filtrated and then DEPC treated and
> >autoclaved.

Why not use a test gel with 1% agarose and denature the RNA for 10 mins @
60C in 3:1 formaldehyde/formamide then run for 1 hour @ 75V.

You should see the bands no problem. PS you could add EtBr to the gell at
the conc below.

Cyber Green is more sensitive. Ask Molecular Probes for freebie. :-)
> 
> Add EtBr to either the sample (as Susan suggested) or to the gel and buffer
> (I use 10 ug per 100 ml).  Staining after running I think is less sensitive
> (most people don't have the patience to stain and de-stain, plus diffusion
> might be a problem).
> 
> >
> >Is the detection level in the range 500 ng or can I see like 20 ng?
> 
> Certainly 500 ng would be pretty easily seen.  20 ng is probably too little
> to see.  I think there are stains that might be more sensitive than EtBr
> though (Cyber green maybe?), if you really need to see less.  Hope this
> helps.
> 
> Mike
Cheers
Bob; Rainy Scotland

-- 
Robert Hartley, Centre for Cell Engineering,Joseph Black Building
University of Glasgow, Glasgow G12 8QQ Tel: ++44 (0)141 330 4756, 
Fax: 0141 330 3730 mailto:rh at mblab.gla.ac.uk
Web : http://www.gla.ac.uk/Inter/CellEngineering






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