danielg@uncmed.med.unc.edu (09/04/90)
Hi netfolk! Time for me to tap into the cumulative intelligence, wisdom, and experience of the net. We are assembling an internal publication (read 'memo') concerning the various problems that can occur in routine Southern blotting. The format for your input is as follows: Anomaly Probable Cause Cure poor signal -poor radionucleotide -troubleshoot labelling procedure incorporation into probe -poor transfer -proper depurination -too little probe -use 2x amt. of probe in random' procedure background in -expired hyb solution -replace lanes Can you help? What problems have you experienced? We started on this quest while being plagued with "Denhardt's spots" on our films. All of the molecular biology gurus around here would say "Well, we get those once in a while, they come and go, we don't know why." Alternatively, they have a 'folk' remedy, something that's supposed to work, but no one is sure why. Other problems we have experienced include: - reverse video effect (lanes are dark, bands are light) - Denhard's spots (spots of radioactivity randomly spread on membrane) - extra bands at top (usu from poor digestion) - degraded DNA - scratches on membrane (poor handling) All comments welcomed, and please include your hunches, there *has* to be logical reasons for these things, incantations don't work!! Thanks, dan __________________________________________________________________________ **** The shallow man has *** | Do not be overly righteous, nor overly **** has opinions; *** | wise: why should you destroy yourself ? **** the deep, *** | - King Solomon, wisest man of his **** convictions. *** | day, Ecclesiastes 7:16.