Northern analysis

Bhuvana Balasubramanian bhuvanab at imgen.bcm.tmc.edu
Tue Jan 3 14:51:36 EST 1995


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.



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