hybridization problems

wetsel_r at wums.wustl.edu wetsel_r at wums.wustl.edu
Mon Oct 5 05:17:57 EST 1992


In a previous article, Klaus.Matthaei at anu.edu.au wrote:
>>To molecular biologists:
>>I am having a problem in probing Northern blot membranes ans slot blot 
>>membranes (Hybond N+). Sllight hazy background is evident on the 
>>membranes, suggesting that the probe is OK.  However, where the RNA is 
>>applied in slot blots or where it has migrated to in Northerns appears 
>>white.  The image looks like a negative.
>>	If you have encountered this problem or if you know of a 
>>possible solution, please leave a message here.
>>Thanks. 
>>Juan
> 
> 
>G'Day Juan
> 
>You have a common problem of not blocking your membrane against
>non-specific hybridisation.  Nylon membrane needs better blocking than
>nitrocellulose.  The negative image is due to the probe not hybridising to
>that part of the membrane and because you have no target = no signal.
> 
>For a homologous DNA probe try hybing in: 50% deionised formamide, 2 x PE,
>7% SDS, 1% BSA (0.5mg/ml Salmon Sperm DNA optional) at 50 degrees.  Wash
>down to 0.2x SSC 1% SDS at 70.
> 
>Good Luck..Klaus
> 
>--------------------------------------------------------------------------
>Catherine Shang
>Gene Targeting
>The John Curtin School of Medical Research
>The Australian National University
>E-mail: Catherine.Shang at anu.edu.au
> 
> 
>--------------------------------------------------------------------------

Greetings... Normally I would agree with Catherine.  However, my wife
and I have experienced this problem in our lab too and it is her impression
that the problem lies with the lot of N+.  We have always used the standard
hybridization solution (5X SSC,, 0.2%Ficoll 400,0.2% BSA, 0.2%PVP,0.5%pyro,
and 1% SDS) ala Maniatis (Salmon sperm optional) and this has worked fine
for us for years...  until we got this one lot of N+ (#13709) and then all
h*ll broke loose.  As initially described, our bands were shadowed and the
background on the blot high.  Only when my wife changed lots of N+ and repeated
the Southern did it work with low background.  

Catherine's advise is sound; however, have you had this problem before with
different lots of N+ or is this a new problem?

Thanks,

David
haviland at kids.wustl.edu



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