Northern blot with uniform high background in slot-containing half
gkidd at aptagen.com
Thu Oct 22 12:19:01 EST 1998
I agree with Ned - the low background in the 26S region is due to the high
amount of 26S RNA swamping the binding sites there. I don't recall ever
getting the other type of background you describe on the upper half of the
blot, so I can't help there. Good luck!
Geoffrey Kidd, Ph.D.
Technical Services Manager
Gerard J.A. Rouwendal wrote:
> Sometimes a puzzling phenomenon occurs with regard to Northern blot
> hybridizations: the slot-containing half of the blot contains a high and
> even background. In addition, the 26S ribosomal RNA appears negative
> (the 18S rRNA is just below the zone with high background).
> Unfortunately, the desired signal is located between these two major
> rRNA bands.
> Details: gel run with glyoxal denatured RNA in 10 mM NaPi buffer using
> buffer circulation; blotted onto Boehringer positively charged membrane
> with NaPi buffer and UV crosslinked; glyoxal removed by boiling in
> Tris-HCl pH 8.0; (pre)hybridization in 0.5 M NaPi pH 7.2, 7% SDS and 2%
> blocking reagent (Boehringer); probe obtained by random primed labeling
> with 32P dCTP of a PCR fragment.
> Does anyone have suggestions as to the cause of this rather peculiar
> type of background?
> Thank you very much in advance,
> Gerard Rouwendal
> +31-317-475118 or 475248
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