Spots on Northern probed with oligos (50-mers)

Andy Phillips andy.phillips at bbsrc.ac.uk
Fri Aug 2 11:09:25 EST 1996


Hi,

We're trying to compare the expression levels of three very 
closely-related homeologous genes in wheat. To get discrimination 
between the transcripts, we're using 50-meric oligos. We're having great 
trouble with background spots all over the filters. We don't see these 
spots with longer (500bp) probes; filtering the probe has no effect.

Conditions:
20% formamide
50mM NaPi pH 6.3
100ug/ml denatured salmon sperm DNA
5 x Denhardts
5 x SSC
0.1% SDS

Hyb'ing at 42oC, on nitrocellulose.

Any suggestions, O wise and thoughful ones?

TIA

Andy
 
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