Northern blot with uniform high background in slot-containing half

John Thompson jrt at home.com
Sat Oct 24 11:19:28 EST 1998


Gerard,
Can't quite tell if this fits the description of your background
problem, but high background over a portion of the blot with a clearly
demarked border is generally due to overlapping of the blot within a
hybridization roller bottle (even when nylon mess is used to
separate).  Use seal-a-meal bags instead.
Regards,
John Thompson

g.j.a.rouwendal at ato.dlo.nl ("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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