southern background problems

Bert Gold gold at astro.ocis.temple.edu
Sun Jun 5 08:36:03 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.

.



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