Weird Northern

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Mon Jul 17 09:30:27 EST 2000


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.

Mike



>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
>rDNAs.
>
>Any suggestion?
>
>Black
>
>
>
>
>---


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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