cxwang at wam.umd.edu
Mon Jul 17 09:46:20 EST 2000
I used the random labeling kit from Boehringer. The hybridization was
performed at 65C and washing condition was high stringent.
On Mon, 17 Jul 2000, Michael L. Sullivan wrote:
> How are you making your probes? In my experience, riboprobes will
> sometimes hybridize (irreversibly-- it doesn't matter how high a stringency
> wash you use) to sequences other than the target unless hybridization is
> carried out at relatively high stringency. This is very probe dependent.
> If you think this might be your problem, you may have to do some empirical
> testing to find an appropriate hybridization condition. Hope this helps.
> >Hi, All:
> >I was doing northern analysis with arabidopsis AOS gene. When I used the
> >5' fragment of the gene as a probe (from the EST), it worked
> >well. However, if I used the 3' fragment as a probe (from PCR), it always
> >hybridized to all the rRNA (including 26S, 18S, 16S) but not the AOS
> >gene. I did three times and got the same result. I sequenced the PCR
> >product and it was correct sequence. then I was suspicous of any homologs
> >there. but the Blast search pulled out no similarity to any other genes or
> >Any suggestion?
> Michael L. Sullivan, Ph.D
> U.S. Dairy Forage Research Center
> 1925 Linden Drive West
> Madison WI, 53706
> (608) 264-5144 Phone
> (608) 264)-5147 Fax
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