In article <36F01CC5.C4F7B077 at agora.ulaval.ca>, Tahar aboulkassim
<abh346 at agora.ulaval.ca> wrote:
>I have always a problem when I do my northern blot experience. The
>problem is the non-specific hybridization of my probes with 28S and 18S
>and I have a strong signal for these RNAs. furthermore, the specific
>signal is either weak or absent. I have changed many parameters in order
>to improve or to resolve this problem. But, always have the same
>It is suspected that the blocking agents may be the source of the
>problem, I then used other blocking agents like poly A+, with those used
>previously e.g. Salmon sperm DNA (Gibco), and Denhardt solution. We used
>several different probes ( DNA and RNA probes) and conditions of
>prehybridization/hybridization : 60°C for RNA probe and 42° for DNA
>probe (with formamide), but the result remained the same.
>>I would be grateful if someone can give me an idea ( may be someone had
>already the same problem) of what could be the problem.
Personally, I don't like to use Denhardt's. I generally get a much better
result with much lower background using Church buffer. For my Ph.D.
thesis, I used it to do dozens upon dozens of Northerns with a cDNA probe
to a low abundance transcription factor using only total, non-poly-A
selected RNA. Church buffer is a pain in the butt to make up, but I
usually make 2 L or so at a time, and that lasts me several months. (I
don't do as many Northerns as I used to do.)
Here's the recipe:
For 500 ml Church buffer
BSA (1%-optional) 5 g
EDTA (1 mM) 1 ml 0.5 M EDTA
NaPO4 (0.5 M, pH-7.5) 33.5 g Na2PO4.7H20 + 1 ml 85% H3PO4
SDS 7% 35 g
Use just like Denhardt's. Wash with various SSC solutions as you normally
would. Salmon sperm DNA is not necessary, although it can be added if you
desire. I don't know if it actually works in Church's to reduce background
since the background is so low to begin with. The beauty of this solution
is that you don't need to prehybridize for a long time. 15 minutes at 65
deg C is more than enough. Even better, if you get greater than 50%
incorporation of 32P into your probe, you don't have to purify the probe
before use in this solution.
Hybridization stringency can be increased by making Church buffer with 15%
formamide and hybridizing at 65 deg C. You can increase stringency still
further by using Church buffer + 15% formamide made with only 0.2 mM NaPO4
and hybridizing at 65 deg C.
This solution will precipitate out if the room gets too cool. Just heat it
to 65 deg C to redissolve.
Orac |"A statement of fact cannot be insolent."--Orac
David Gorski |"If you cannot listen to the answers, why do you
U. of Chicago| inconvenience me with questions?"--Orac again