Southern problems

Joseph C. Bagshaw jbagshaw at wpi.edu
Tue Jul 7 16:27:17 EST 1998


 You might try substituting the primers used to PCR the fragment for the
random primers.  However, it sounds like your fighting the never-ending
battle for truth, justice, and low-copy-number signals.  With a
whole-plasmid probe, every band in Brl's ladder will light up like
gangbusters.  I routinely cut the marker lane off even before blotting,
although this will not cure your sensitivity problem except that you can
leave the blot on the film for a long exposure.  You might also try a
biotin-labelled probe; they really are more sensitive IF you use the right
kit.  (Oops, did I just start another kit flame war?)

********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.edu
Roadkill on the information superhighway.

On Fri, 3 Jul 1998 sarah_petersen at yahoo.com wrote:

> I'd like some advice:
> 
> I'm trying to detect a low-copy number sequence in a genomic DNA Southern.
> The genome size is ~100Mb, and I've loaded 7-10 micrograms of RE-digested DNA
> per lane on nylon. The probe is a cloned fragment ~300bp long which I have
> prepared by PCR-ing the fragment out of its vector (using fragment-specific,
> not vector-specific, primers), ppting with EtOH, resuspending in HOH, and
> random primed labeling with 32P (using B-M High Prime) - used about 50-100ng
> for labeling. I filtered the probe through a G-50 gravity column, and hybed
> O/N in a 7% SDS phosphate buffer. The probe (eluted from the G-50 column) was
> in about 1/2 ml of TE, and was added to 3 mls of the hyb. solution (hyb in
> roller bottle). The membrane was about 5X10 CM (5 lanes). Hybed O/N at 60C,
> washed 2X5 min at RT, 2XSSPE, 0.1%SDS, 3X5 min at 60C, 1XSSPE, 0.1%SDS. On
> film at -80 with intensifying screen O/N. Before putting the blot to film, I
> had to cut off the two marker lanes (BRL 1Kb+) because the signal was too hot
> there. I left one of the cut-out lanes on the film (away from the genomic
> blot).
> 
> Results? The genomic part of the film was blank. Little noise, no signal. The
> cut-off marker lane showed *every* band. I guess I overloaded it but didn't
> think I'd have enough vector stuff labeled to show it up that brightly (it is
> vector bits that light that up, isn't it?)
> 
> Anyway, I'm thinking that 300bp must be marginal for random-primed labeling
> and that I'd be better off end-labeling, or treating the random-primed probe
> as an oligo and lowering the hyb. temp to 50 or so. But perhaps I'm missing
> something fundamental here?
> 
> Thanks for any suggestions, especially the *good* ones :-)
> 
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