Weird Northern again!!!

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Mon Jul 24 09:03:36 EST 2000


Hmmm.  I'm not sure what'g oing on here.

Have you done your EST probe Northern as a control at the same time with
the same buffers as the PCR probed northerns?  If not, try that and see if
it is now problematic too.  If it is, perhaps something has changed, such
as your hybridization buffer or even the membrane.

Hope this helps.

Mike

PS-- Sometimes when something like this is going on, it's useful if you can
talk a collegue into running and hybridizing a blot for you.


>This time two new probes PCR-amplified from arabidopsis PR-1 and VSP1
>genes, repectively, were used and the membrane was newly transferred. I
>did the hybridization at the same time in different bags. However, the
>result was the same: all signals came from the rRNAs. The hybridization
>pattern was exactly the same as the EB-stained gel pattern.
>
>why it happened to me so often in so short time?I was so desperate now.
>
>Black
>
>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.
>>
>> 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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