Reason for non-specific bands from Northern?

Vladimir Grubor vgrubor at pgrad.unimelb.edu.au
Thu Jan 31 23:54:52 EST 2002


Hi there,

How did you label the probe? With a hot PCR or with random labeling (Klenow 
fragment?). If hot PCR, your probe could be too large and will 
cross-hybridise to a non-specific RNA. Also what was the stringency of your 
washes? Make sure that you are making them highly stringent and that you 
use lots of blocking agent (i.e. Salmon sperm/testes ssDNA or 7M SDS).

Hope that this helps.

Regards,

Vladimir Grubor


Mengjuan Guo wrote:

> I just did Northern on an Arabidopsis gene with Ultrahyb buffer from
> Ambion, but got mutiple bands after 15 hours hybridization.  I think my
> probe was specific, because when BLAST the nucleotide sepquence of this
> probe, no homology was found in Arabidopsis genome. So what could be the
> problem? Could the Ultrahyb be too sensitive? Thanks for help!




More information about the Methods mailing list