southern background problems

Lauri Lintott llintott at ACS.UCALGARY.CA
Mon Jun 6 12:49:05 EST 1994


> 
> Occasionally, I have problems with high backgrounds on mammalian
> genomic DNAs probed with 32P random oligo primed cloned isolated
> fragments.  My post-label clean-up is Gene Clean.  My filter is
> Nytran.  My first washes are 2X SET (0.3M NaCl, 60 mM Tris-HCl pH 8.00,
> 2 mM EDTA), 0.5% SDS at 68 degrees C.  My final wash is 0.1X SET, 0.1%
> SDS at 68 degrees C.  My hybridization conditions are 5X SET, 0.5% SDS,
> 1X Denhardt's and 10% DEXTRAN SULFATE.  This works very well 4 out of
> 5 times, but the fifth time is a bummer.  My geiger counter is giving
> me 1000-4000 cpms from the blot at this moment, and alot of that is
> from the edges of the NYTRAN where I know that there was no DNA.
> There is some chance that the blot short-circuited during blotting,
> but if the DNA were shmeared, I shouln't be able to see remnants of
> nice-looking Ethidium Bromide stained lanes (which I can) when I put
> the hot gel on the transilluminator. (Incidentally, no big need for
> radioactive clean-up on the transilluminar) because the filter (I mistakenly)
> said gel in the sentence above, is not leaching any of its counts.
> The only thing I can think at this point, after about 12 hours of final
> wash is that it might be RNA?  So I am treating the filter with 1 ug/ml
> of ribonuclease in final wash solution at 37 degrees C.
> 
> Any suggestions, netters?
> 
> Bert Gold,
> presently sitting at Jefferson Medical College,
> Philadelphia.
> 
> 
Dear Bert,
I use a different membrane from you and also different wash
buffer (Hybond N+ and SSC buffer) but I would guess that the
problem is in your prehyb which you didn't describe.  It's been
awhile since I made my own prehyb (I use Amersham's rapid hyb now
and it works wounders) but I found that including sonicated Salmon sperm
DNA (1 in 500 of a 10 mg/ml solution) in the prehyb prevented the
type of background problem you describe.  If you are hybridizing
in bags any large air bubbles can interfer with the prehyb.  I
have also heard that increasing the SDS concentration in
hybridization buffer, to 1 % or so can help but I have never
tried that.
Hope I was of some help.  If you need more details I will happily
provide them.

Lauri
Biological Sciences
U of Calgary

































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