Southern blot QC and Various Anomalies

danielg at uncmed.med.unc.edu danielg at uncmed.med.unc.edu
Tue Sep 4 07:35:02 EST 1990


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.



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