HELP! Lane background in Northern's

[Lauri Lintott]

Gil (kgilbert at cmp.hmc.psu.edu) wrote:
: Here are some more specifics  which may help someone with my background
: problem:

: I run a 1.2% agarose/0.4M formaldehyde gel; downward capillary transfer
: in 10X SSPE overnight; membrane is washed in 5X SSPE @ 60 degrees for 5
: min, then baked @80 degrees for 1 hr, and UV crosslinked in a
: Stratalinker (200 ujX100).  

: After O/N hybridization, washes are as follows:
: 1. Quick rinse in 2X SSPE
: 2. 2 x 15 min washes in 2XSSPE/0.5%SDS at hybridization temperature


Dear Gil,
Try washing with higher stringency.  High background in the lanes
only suggests your probe is hybridizing to everything.  For
Northerns with a specific probe I usually was in 0.1 X SSC/0.5 %
SDS at 60 - 65 C, 2 X 30 min.  Just keep raising your stringency
until the X-ray exposures look good.