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Refined protocol

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The Roche/BM protocol is not ideal. The blocking reagent readily precipitates and gives high background, and the detection buffer is less than ideal due to a low salt concentration. In a paper 'Detection of Single-Copy Nonradioactive Southern Blot', Matthew McCabe et al describe a much improved protocol based upon past publications and new refinements. I would recommend anyone about to carry out the DIG Southern for eukaryotic organisms, or DIG Northern for anything other than highly expressed genes, or just wanting a clean and sensitive that paper!!!

Broadly, the detection buffer needs to be changed to 3M NaCl 100mM maleic acid pH 8 (free) autoclaved, then made 0.3% with Tween 20 before use...and the blocker needs to be miracloth filtered before autoclaving, after having been CAREFULLY pHd to 7.5....while the Antibody solution should be used at 1:10000. These and other changes, including washing the blot after the post hyb hot wash with the detection buffer to remove SDS before attempting the Western blocking step...should give extra sensitive results...

Jon Rees

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