Southern Blot Analysis

heather hmitchel at BU.EDU
Mon Jun 8 14:31:17 EST 1998


Hi all,

I have recently been experiencing strange background problems on
Southern blots I have been performing.  The autorads show high
background all around the perimeter of the blot and splotches of
radioactivity (large and small) randomly arranged around the center of
the blot.  The signal is visible in areas where the splotches are not. 
In addition the top 2/3 of the blot often has HIGH background while the
bottom 1/3 remains fairly clean.  I was wondering if anyone else has had
similar problems and if so what do I need to do to fix it.  Here are the
specifics:

after running 20ug total muscle DNA/lane on 1% agarose gels, I incubate
in 0.25N HCl and then 0.4M NaOH, followed by transfer of the DNA onto
HybondN+ membrane using an alkaline downward transfer method.  I wash
the blots briefly in 2xSSC.  Then I place them in glass hybridization
bottles  with pre-hyb solution, pre-hyb at 68 C for 4-20 hours, and then
add a random primed probe and incubate overnight at 68 C.  My most
stringent wash is 0.1xSSC/0.1%SDS at 42 C for a half hour.  Oh - I can
wash the probe off the target before the background splotches disappear
with higher stringencies.

The first type of pre-hyb soln I tried contained 5x SSC, 5x Denhardts,
0.1% SDS and denatured calf thymus DNA (100ug/ml).  I then tried a
pre-hyb soln with 5x SSPE, 5x Denhardts, 0.1% SDS, 0.5% NaPPi, and
denatured calf thymus DNA (100ug/ml).  I used 2mls pre-hyb/hyb per 1cm^2
membrane surface.  Both pre-hyb/hyb solns give the same type of
background though the SSPE mix is not quite as bad as the SSC.

I also have experimented with removing the mesh screens we historically
use in the hyb. bottles.  Other than an overall lowering of the signal
(and background) I can not see any difference with or without the
meshes.

Would using a different pre-hyb buffer work better (e.g. Church's
soln).  Would using salmon sprem DNA instead of calf thymus DNA be
better???  I ahve read that some pre-hyb solns contain
N-laurylsarcosine, or EDTA,....  Would these help prevent the
non-specific background I am seeing.

Any advice would be greatly appreciated.  Please e-mail me with any
suggestions (hmitchel at bu.edu).

Thanks a BUNCH :-)
heather mitchell-felton



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