Southern problems

sarah_petersen at yahoo.com sarah_petersen at yahoo.com
Fri Jul 3 16:56:08 EST 1998


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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