Northern analysis

[John Aitchison]

In article <3ec9s8$ihb at gazette.bcm.tmc.edu>, bhuvanab at imgen.bcm.tmc.edu
(Bhuvana Balasubramanian) wrote:

> Hi, 
>      Does anybody know the sizes of the two rRNAS (yeast)which appear as
> prominent bands on a northern blot?I think they are 28S(?) and 18S(~1.9kb?) 
>      I have a 2.7kb ORF whose transcript runs well above these two rRNAs.I am
> trying to estimate the size of this without RNA markers, but I think I am
> having trouble with transfering RNA above the rRNAs from the gel.Total
> cellular RNA is extracted from yeast using the glass beads/phenol protocol
> and I run 20ug of it on a 1% formaldehyde gel.I treat the gel with
> .05NNaOH/.15N NaCl and .15N NaCl/10mM Tris-pH7.6 prior to the transfer.I 
> stain the gel with EtBr.The transfer is done at 4deg. C o/n.I have seen a
> signal well above the rRNA bands upon probing the blot with a 2kb fragment
> within the gene.But I am having trouble reproducing my preliminary look-see.
> Any suggestions would be helpful.Thanks.bhuvana.

Bhuvana:  This looks much like the protocol from "The Red Book"    (Short
protocols in Mol Biol. by Ausubel et al.)  The sizes of the ribosomal rnas
are in this book.  I dont think there should be any trouble with this
procedure.  Is the RNA getting degraded?  This is likely a problem if you
saw it proviously, but no longer.  I dont think it is necessary to
transfer in the cold and etbr staining is not necessary.  If you transfer
to nylon and just hold the membrane to a UV box (on its side) you will be
able to see the ribosomal rna markers.   I guess it is stating the
obvious, but transferring longer should help if the RNA is intact.  
John

John Aitchison                      212 327-8099 (lab)
Laboratory of Cell Biology           212 327-8660 (fax)
The Rockefeller University/HHMI
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