hybridization problems

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Wed Sep 30 18:30:22 EST 1992

>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.

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


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